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Francesca FANELLI
Professore Ordinario
Dipartimento di Scienze della Vita sede ex Chimica V.Campi 103
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Pubblicazioni
2023
- Wordom update 2: A user-friendly program for the analysis of molecular structures and conformational ensembles
[Articolo su rivista]
Felline, A.; Conti, S.; Seeber, M.; Cecchini, M.; Fanelli, F.
abstract
We present the second update of Wordom, a user-friendly and efficient program for manipulation and analysis of conformational ensembles from molecular simulations. The actual update expands some of the existing modules and adds 21 new modules to the update 1 published in 2011. The new adds can be divided into three sets that: 1) analyze atomic fluctuations and structural communication; 2) explore ion-channel conformational dynamics and ionic translocation; and 3) compute geometrical indices of structural deformation. Set 1 serves to compute correlations of motions, find geometrically stable domains, identify a dynamically invariant core, find changes in domain-domain separation and mutual orientation, perform wavelet analysis of large-scale simulations, process the output of principal component analysis of atomic fluctuations, perform functional mode analysis, infer regions of mechanical rigidity, analyze overall fluctuations, and perform the perturbation response scanning. Set 2 includes modules specific for ion channels, which serve to monitor the pore radius as well as water or ion fluxes, and measure functional collective motions like receptor twisting or tilting angles. Finally, set 3 includes tools to monitor structural deformations by computing angles, perimeter, area, volume, β-sheet curvature, radial distribution function, and center of mass. The ring perception module is also included, helpful to monitor supramolecular self-assemblies. This update places Wordom among the most suitable, complete, user-friendly, and efficient software for the analysis of biomolecular simulations. The source code of Wordom and the relative documentation are available under the GNU general public license at http://wordom.sf.net.
2023
- psnGPCRdb: The Structure-network Database of G Protein Coupled Receptors
[Articolo su rivista]
Felline, A.; Gentile, S.; Fanelli, F.
abstract
G protein coupled receptors (GPCRs) are critical eukaryotic signal transduction gatekeepers and represent the largest protein superfamily in the human proteome, with more than 800 members. They share seven transmembrane helices organized in an up-down bundle architecture. GPCR-mediated signaling pathways have been linked to numerous human diseases, and GPCRs are the targets of approximately 35% of all drugs currently on the market. Structure network analysis, a graph theory-based approach, represents a cutting-edge tool to deeply understand GPCR function, which strongly relies on communication between the extracellular and intracellular poles of their structure. psnGPCRdb stores the structure networks (i.e., linked nodes, hubs, communities and communication pathways) computed on all updated GPCR structures in the Protein Data Bank, in their isolated states or in complex with extracellular and/or intracellular molecules. The structure communication signatures of a sub-family or family of GPCRs as well as of their small-molecule activators or inhibitors are stored as consensus networks. The database stores also all meaningful structure network-based comparisons (i.e., difference networks) of functionally different states (i.e., inactive or active) of a given receptor sub-type, or of consensus networks representative of a receptor sub-type, type, sub-family or family. Single or consensus GPCR networks hold also information on amino acid conservation. The database allows to graphically analyze 3D structure networks together with interactive data-tables. Ligand-centric networks can be analyzed as well. psnGPCRdb is unique and represents a powerful resource to unravel GPCR function with important implications in cell signaling and drug design. psnGPCRdb is freely available at: http://webpsn.hpc.unimo.it/psngpcr.php.
2022
- Functional rewiring of G protein-coupled receptor signaling in human labor
[Articolo su rivista]
Walker, A. R.; Larsen, C. B.; Kundu, S.; Stavrinidis, C.; Kim, S. H.; Inoue, A.; Woodward, D. F.; Lee, Y. S.; Migale, R.; Macintyre, D. A.; Terzidou, V.; Fanelli, F.; Khanjani, S.; Bennett, P. R.; Hanyaloglu, A. C.
abstract
Current strategies to manage preterm labor center around inhibition of uterine myometrial contractions, yet do not improve neonatal outcomes as they do not address activation of inflammation. Here, we identify that during human labor, activated oxytocin receptor (OTR) reprograms the prostaglandin E2 receptor, EP2, in the pregnant myometrium to suppress relaxatory/Gαs-cAMP signaling and promote pro-labor/inflammatory responses via altered coupling of EP2 from Gαq/11 to Gαi/o. The ability of EP2 to signal via Gαi/o is recapitulated with in vitro OT and only following OTR activation, suggesting direct EP2-OTR crosstalk. Super-resolution imaging with computational modeling reveals OT-dependent reorganization of EP2-OTR complexes to favor conformations for Gαi over Gαs activation. A selective EP2 ligand, PGN9856i, activates the relaxatory/Gαs-cAMP pathway but not the pro-labor/inflammatory responses in term-pregnant myometrium, even following OT. Our study reveals a mechanism, and provides a potential therapeutic solution, whereby EP2-OTR functional associations could be exploited to delay preterm labor.
2022
- PSNtools for standalone and web-based structure network analyses of conformational ensembles
[Articolo su rivista]
Felline, A.; Seeber, M.; Fanelli, F.
abstract
Structure graphs, in which interacting amino acids/nucleotides correspond to linked nodes, represent cutting-edge tools to investigate macromolecular function. The graph-based approach defined as Protein Structure Network (PSN) was initially implemented in the Wordom software and subsequently in the webPSN server. PSNs are computed either on a molecular dynamics (MD) trajectory (PSN-MD) or on a single structure. In the latter case, information on atomic fluctuations is inferred from the Elastic Network Model-Normal Mode Analysis (ENM-NMA) (PSN-ENM). While Wordom performs both PSN-ENM and PSN-MD analyses but without output post-processing, the webPSN server performs only single-structure PSN-EMN but assisting the user in input setup and output analysis. Here we release for the first time the standalone software PSNtools, which allows calculation and post-processing of PSN analyses carried out either on single structures or on conformational ensembles. Relevant unique and novel features of PSNtools are either comparisons of two networks or computations of consensus networks on sets of homologous/analogous macromolecular structures or conformational ensembles. Network comparisons and consensus serve to infer differences in functionally different states of the same system or network-based signatures in groups of bio-macromolecules sharing either the same functionality or the same fold. In addition to the new software, here we release also an updated version of the webPSN server, which allows performing an interactive graphical analysis of PSN-MD, following the upload of the PSNtools output. PSNtools, the auxiliary binary version of Wordom software, and the WebPSN server are freely available at http://webpsn.hpc.unimo.it/wpsn3.php.
2022
- Structural communication between the GTPase Sec4p and its activator Sec2p: Determinants of GEF activity and early deformations to nucleotide release
[Articolo su rivista]
Felline, A.; Raimondi, F.; Gentile, S.; Fanelli, F.
abstract
Ras GTPases are molecular switches that cycle between OFF and ON states depending on the bound nucleotide (i.e. GDP-bound and GTP-bound, respectively). The Rab GTPase, Sec4p, plays regulatory roles in multiple steps of intracellular vesicle trafficking. Nucleotide release is catalyzed by the Guanine Nucleotide Exchange Factor (GEF) Sec2p. Here, the integration of structural information with molecular dynamics (MD) simulations addressed a number of questions concerning the intrinsic and stimulated dynamics of Sec2p and Sec4p as well as the chain of structural deformations leading to GEF-assisted activation of the Rab GTPase. Sec2p holds an intrinsic ability to adopt the conformation found in the crystallographic complexes with Sec4p, thus suggesting that the latter selects and shifts the conformational equilibrium towards a pre-existing bound-like conformation of Sec2p. The anchoring of Sec4p to a suitable conformation of Sec2p favors the Sec2p-assisted pulling on itself of the α1/switch 1 (SWI) loop and of SWI, which loose any contact with GDP. Those deformations of Sec4p would occur earlier. Formation of the final Sec2p-Sec4p hydrophobic interface, accomplishes later. Disruption of the nucleotide cage would cause firstly loss of interactions with the guanine ring and secondly loss of interactions with the phosphates. The ease in sampling the energy landscape and adopting a bound-like conformation likely favors the catalyzing ability of GEFs for Ras GTPases.
2021
- Frontiers in Multiscale Modeling of Photoreceptor Proteins
[Articolo su rivista]
Mroginski, M. -A.; Adam, S.; Amoyal, G. S.; Barnoy, A.; Bondar, A. -N.; Borin, V. A.; Church, J. R.; Domratcheva, T.; Ensing, B.; Fanelli, F.; Ferre, N.; Filiba, O.; Pedraza-Gonzalez, L.; Gonzalez, R.; Gonzalez-Espinoza, C. E.; Kar, R. K.; Kemmler, L.; Kim, S. S.; Kongsted, J.; Krylov, A. I.; Lahav, Y.; Lazaratos, M.; Nassereddin, Q.; Navizet, I.; Nemukhin, A.; Olivucci, M.; Olsen, J. M. H.; Perez de Alba Ortiz, A.; Pieri, E.; Rao, A. G.; Rhee, Y. M.; Ricardi, N.; Sen, S.; Solov'Yov, I. A.; De Vico, L.; Wesolowski, T. A.; Wiebeler, C.; Yang, X.; Schapiro, I.
abstract
This perspective article highlights the challenges in the theoretical description of photoreceptor proteins using multiscale modeling, as discussed at the CECAM workshop in Tel Aviv, Israel. The participants have identified grand challenges and discussed the development of new tools to address them. Recent progress in understanding representative proteins such as green fluorescent protein, photoactive yellow protein, phytochrome, and rhodopsin is presented, along with methodological developments.
2021
- Structural aspects of rod opsin and their implication in genetic diseases
[Articolo su rivista]
Fanelli, F.; Felline, A.; Marigo, V.
abstract
Vision in dim-light conditions is triggered by photoactivation of rhodopsin, the visual pigment of rod photoreceptor cells. Rhodopsin is made of a protein, the G protein coupled receptor (GPCR) opsin, and the chromophore 11-cis-retinal. Vertebrate rod opsin is the GPCR best characterized at the atomic level of detail. Since the release of the first crystal structure 20 years ago, a huge number of structures have been released that, in combination with valuable spectroscopic determinations, unveiled most aspects of the photobleaching process. A number of spontaneous mutations of rod opsin have been found linked to vision-impairing diseases like autosomal dominant or autosomal recessive retinitis pigmentosa (adRP or arRP, respectively) and autosomal congenital stationary night blindness (adCSNB). While adCSNB is mainly caused by constitutive activation of rod opsin, RP shows more variegate determinants affecting different aspects of rod opsin function. The vast majority of missense rod opsin mutations affects folding and trafficking and is linked to adRP, an incurable disease that awaits light on its molecular structure determinants. This review article summarizes all major structural information available on vertebrate rod opsin conformational states and the insights gained so far into the structural determinants of adCSNB and adRP linked to rod opsin mutations. Strategies to design small chaperones with therapeutic potential for selected adRP rod opsin mutants will be discussed as well.
2021
- Structure network-based landscape of rhodopsin misfolding by mutations and algorithmic prediction of small chaperone action
[Articolo su rivista]
Felline, A.; Schiroli, D.; Comitato, A.; Marigo, V.; Fanelli, F.
abstract
Failure of a protein to achieve its functional structural state and normal cellular location contributes to the etiology and pathology of heritable human conformational diseases. The autosomal dominant form of retinitis pigmentosa (adRP) is an incurable blindness largely linked to mutations of the membrane protein rod opsin. While the mechanisms underlying the noxious effects of the mutated protein are not completely understood, a common feature is the functional protein conformational loss. Here, the wild type and 39 adRP rod opsin mutants were subjected to mechanical unfolding simulations coupled to the graph theory-based protein structure network analysis. A robust computational model was inferred and in vitro validated in its ability to predict endoplasmic reticulum retention of adRP mutants, a feature linked to the mutation-caused misfolding. The structure-based approach could also infer the structural determinants of small chaperone action on misfolded protein mutants with therapeutic implications. The approach is exportable to conformational diseases linked to missense mutations in any membrane protein.
2020
- Dynamics and structural communication in the ternary complex of fully phosphorylated V2 vasopressin receptor, vasopressin, and β-arrestin 1
[Articolo su rivista]
Bellucci, L.; Felline, A.; Fanelli, F.
abstract
G protein-coupled receptors (GPCRs) are critically regulated by arrestins, which not only desensitize G-protein signaling but also initiate a G protein-independent wave of signaling. The information from structure determination was herein exploited to build a structural model of the ternary complex, comprising fully phosphorylated V2 vasopressin receptor (V2R), the agonist arginine vasopressin (AVP), and β-arrestin 1 (β-arr1). Molecular simulations served to explore dynamics and structural communication in the ternary complex. Flexibility and mechanical profiles reflect fold of V2R and β-arr1. Highly conserved amino acids tend to behave as hubs in the structure network and contribute the most to the mechanical rigidity of V2R seven-helix bundle and of β-arr1. Two structurally and dynamically distinct receptor-arrestin interfaces assist the twist of the N- and C-terminal domains (ND and CD, respectively) of β-arr1 with respect to each other, which is linked to arrestin activation. While motion of the ND is essentially assisted by the fully phosphorylated C-tail of V2R (V2RCt), that of CD is assisted by the second and third intracellular loops and the cytosolic extensions of helices 5 and 6. In the presence of the receptor, the β-arr1 inter-domain twist angle correlates with the modes describing the essential subspace of the ternary complex. β-arr1 motions are also influenced by the anchoring to the membrane of the C-edge-loops in the β-arr1-CD. Overall fluctuations reveal a coupling between motions of the agonist binding site and of β-arr1-ND, which are in allosteric communication between each other. Mechanical rigidity points, often acting as hubs in the structure network and distributed along the main axis of the receptor helix bundle, contribute to establish a preferential communication pathway between agonist ligand and the ND of arrestin. Such communication, mediated by highly conserved amino acids, involves also the first amino acid in the arrestin C-tail, which is highly dynamic and is involved in clathrin-mediated GPCR internalization.
2020
- ETNK1 mutations in atypical chronic myeloid leukemia induce a mutator phenotype that can be reverted with phosphoethanolamine
[Articolo su rivista]
Fontana, D.; Mauri, M.; Renso, R.; Docci, M.; Crespiatico, I.; Rost, L. M.; Jang, M.; Niro, A.; D'Aliberti, D.; Massimino, L.; Bertagna, M.; Zambrotta, G.; Bossi, M.; Citterio, S.; Crescenzi, B.; Fanelli, F.; Cassina, V.; Corti, R.; Salerno, D.; Nardo, L.; Chinello, C.; Mantegazza, F.; Mecucci, C.; Magni, F.; Cavaletti, G.; Bruheim, P.; Rea, D.; Larsen, S.; Piazza, R.; Gambacorti-Passerini, C.
abstract
2020
- ETNK1 mutations induce a mutator phenotype that can be reverted with phosphoethanolamine
[Articolo su rivista]
Fontana, D.; Mauri, M.; Renso, R.; Docci, M.; Crespiatico, I.; Rost, L. M.; Jang, M.; Niro, A.; D'Aliberti, D.; Massimino, L.; Bertagna, M.; Zambrotta, G.; Bossi, M.; Citterio, S.; Crescenzi, B.; Fanelli, F.; Cassina, V.; Corti, R.; Salerno, D.; Nardo, L.; Chinello, C.; Mantegazza, F.; Mecucci, C.; Magni, F.; Cavaletti, G.; Bruheim, P.; Rea, D.; Larsen, S.; Gambacorti-Passerini, C.; Piazza, R.
abstract
Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype.
2020
- Integrated structural modeling and super-resolution imaging resolve GPCR oligomers
[Articolo su rivista]
Fanelli, F.; Hanyaloglu, A. C.; Jonas, K.
abstract
Formation of G protein-coupled receptors (GPCRs) dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. For the Class A/rhodopsin subfamily of glycoprotein hormone receptors (GpHRs), di/oligomerization has been demonstrated to play a significant role in regulating its signaling activity at a cellular and physiological level and even pathophysiologically. Here we will describe and discuss the developments in our understanding of GPCR oligomerization, in both health and disease, from the study of this unique and complex subfamily of GPCRs with light on the luteinizing hormone receptor (LHR). Focus will be put on the results of an approach relying on the combination of atomistic modeling by protein-protein docking with super-resolution imaging. The latter could resolve single LHR molecules to ~ 8 nm resolution in functional asymmetric dimers and oligomers, using dual-color photoactivatable dyes and localization microscopy (PD-PALM). Structural modeling of functionally asymmetric LHR trimers and tetramers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.
2020
- Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival
[Articolo su rivista]
Casarini, L.; Lazzaretti, C.; Paradiso, E.; Limoncella, S.; Riccetti, L.; Sperduti, S.; Melli, B.; Marcozzi, S.; Anzivino, C.; Sayers, N. S.; Czapinski, J.; Brigante, G.; Poti, F.; La Marca, A.; De Pascali, F.; Reiter, E.; Falbo, A.; Daolio, J.; Villani, M. T.; Lispi, M.; Orlando, G.; Klinger, F. G.; Fanelli, F.; Rivero-Muller, A.; Hanyaloglu, A. C.; Simoni, M.
abstract
Molecular Biology; Female Reproductive Endocrinology; Endocrine Regulation
2020
- webPSN v2.0: a webserver to infer fingerprints of structural communication in biomacromolecules
[Articolo su rivista]
Felline, A.; Seeber, M.; Fanelli, F.
abstract
A mixed Protein Structure Network (PSN) and Elastic Network Model-Normal Mode Analysis (ENM-NMA)-based strategy (i.e. PSN-ENM) was developed to investigate structural communication in bio-macromolecules. Protein Structure Graphs (PSGs) are computed on a single structure, whereas information on system dynamics is supplied by ENM-NMA. The approach was implemented in a webserver (webPSN), which was significantly updated herein. The webserver now handles both proteins and nucleic acids and relies on an internal upgradable database of network parameters for ions and small molecules in all PDB structures. Apart from the radical restyle of the server and some changes in the calculation setup, other major novelties concern the possibility to: a) compute the differences in nodes, links, and communication pathways between two structures (i.e. network difference) and b) infer links, hubs, communities, and metapaths from consensus networks computed on a number of structures. These new features are useful to identify commonalties and differences between two different functional states of the same system or structural-communication signatures in homologous or analogous systems. The output analysis relies on 3D-representations, interactive tables and graphs, also available for download. Speed and accuracy make this server suitable to comparatively investigate structural communication in large sets of bio-macromolecular systems. URL: http://webpsn.hpc.unimore.it.
2019
- A-ARM: Automatic Rhodopsin Modeling with Chromophore Cavity Generation, Ionization State Selection, and External Counterion Placement
[Articolo su rivista]
Pedraza-Gonzalez, L.; De Vico, L.; Marln, M. D. C.; Fanelli, F.; Olivucci, M.
abstract
The Automatic Rhodopsin Modeling (ARM) protocol has recently been proposed as a tool for the fast and parallel generation of basic hybrid quantum mechanics/molecular mechanics (QM/MM) models of wild type and mutant rhodopsins. However, in its present version, input preparation requires a few hours long user's manipulation of the template protein structure, which also impairs the reproducibility of the generated models. This limitation, which makes model building semiautomatic rather than fully automatic, comprises four tasks: definition of the retinal chromophore cavity, assignment of protonation states of the ionizable residues, neutralization of the protein with external counterions, and finally congruous generation of single or multiple mutations. In this work, we show that the automation of the original ARM protocol can be extended to a level suitable for performing the above tasks without user's manipulation and with an input preparation time of minutes. The new protocol, called a-ARM, delivers fully reproducible (i.e., user independent) rhodopsin QM/MM models as well as an improved model quality. More specifically, we show that the trend in vertical excitation energies observed for a set of 25 wild type and 14 mutant rhodopsins is predicted by the new protocol better than when using the original. Such an agreement is reflected by an estimated (relative to the probed set) trend deviation of 0.7 ± 0.5 kcal mol -1 (0.03 ± 0.02 eV) and mean absolute error of 1.0 kcal mol -1 (0.04 eV).
2019
- Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum
[Articolo su rivista]
Subramanian, Advait; Capalbo, Anita; Iyengar, Namrata Ravi; Rizzo, Riccardo; di Campli, Antonella; Di Martino, Rosaria; Lo Monte, Matteo; Beccari, Andrea R.; Yerudkar, Amol; del Vecchio, Carmen; Glielmo, Luigi; Turacchio, Gabriele; Pirozzi, Marinella; Kim, Sang Geon; Henklein, Petra; Cancino, Jorge; Parashuraman, Seetharaman; Diviani, Dario; Fanelli, Francesca; Sallese, Michele; Luini, Alberto
abstract
Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction. By sensing the load of folded ER lumenal proteins, the COPII subunit Sec24 directs a signaling cascade that allows secretory pathway flux to respond to the abundance of cargo.
2019
- Frontal Variant of Alzheimer's Disease: A Report of a Novel PSEN1 Mutation
[Articolo su rivista]
Monacelli, F.; Martella, L.; Parodi, M. N.; Odetti, P.; Fanelli, F.; Tabaton, M.
abstract
Alzheimer's disease may mimic frontotemporal dementia. We describe a case of presenile dementia who presented with peudo-psychotic symptoms carrying a PSEN1 mutation (P355S), which was not known to be pathogenic. PET-FDG showed bilateral frontotemporal hypometabolism, but at MRI, multiple microbleeds were detected, suggestive of amyloid angiopathy.
2019
- Interconnecting Flexibility, Structural Communication, and Function in RhoGEF Oncoproteins
[Articolo su rivista]
Felline, A.; Belmonte, L.; Raimondi, F.; Bellucci, L.; Fanelli, F.
abstract
Dbl family Rho guanine nucleotide exchange factors (RhoGEFs) play a central role in cell biology by catalyzing the exchange of guanosine 5′-triphosphate for guanosine 5′-diphosphate (GDP) on RhoA. Insights into the oncogenic constitutive activity of the Lbc RhoGEF were gained by analyzing the structure and dynamics of the protein in different functional states and in comparison with a close homologue, leukemia-associated RhoGEF. Higher intrinsic flexibility, less dense and extended structure network, and less stable allosteric communication pathways in Lbc, compared to the nonconstitutively active homologue, emerged as major determinants of the constitutive activity. Independent of the state, the essential dynamics of the two RhoGEFs is contributed by the last 10 amino acids of Dbl homology (DH) and the whole pleckstrin homology (PH) domains and tends to be equalized by the presence of RhoA. The catalytic activity of the RhoGEF relies on the scaffolding action of the DH domain that primarily turns the switch I (SWI) of RhoA on itself through highly conserved amino acids participating in the stability core and essential for function. Changes in the conformation of SWI and disorganization of the RhoA regions deputed to nucleotide binding are among the major RhoGEF effects leading to GDP release. Binding of RhoA reorganizes the allosteric communication on RhoGEF, strengthening the communication among the canonical RhoA binding site on DH, a secondary RhoA binding site on PH, and the binding site for heterotrimeric G proteins, suggesting dual roles for RhoA as a catalysis substrate and as a regulatory protein. The structure network-based analysis tool employed in this study proved to be useful for predicting potentially druggable regulatory sites in protein structures.
2018
- A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness
[Articolo su rivista]
Behnen, Petra; Felline, Angelo; Comitato, Antonella; Di Salvo, Maria Teresa; Raimondi, Francesco; Gulati, Sahil; Kahremany, Shirin; Palczewski, Krzysztof; Marigo, Valeria; Fanelli, Francesca
abstract
The autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants.
2018
- Computational modeling approaches to quantitative structure–binding kinetics relationships in drug discovery
[Articolo su rivista]
De Benedetti, Pier G.; Fanelli, Francesca
abstract
Simple comparative correlation analyses and quantitative structure–kinetics relationship (QSKR) models highlight the interplay of kinetic rates and binding affinity as an essential feature in drug design and discovery. The choice of the molecular series, and their structural variations, used in QSKR modeling is fundamental to understanding the mechanistic implications of ligand and/or drug–target binding and/or unbinding processes. Here, we discuss the implications of linear correlations between kinetic rates and binding affinity constants and the relevance of the computational approaches to QSKR modeling.
2017
- Class A GPCR: Di/Oligomerization of Glycoprotein Hormone Receptors
[Capitolo/Saggio]
Hanyaloglu, A. C.; Fanelli, F.; Jonas, K. C.
abstract
G protein-coupled receptor (GPCR) dimerization and oligomerization was first described over 2 decades ago, contributing to the recent paradigm shift in GPCR signaling of a simplistic, archetypal view involving single receptors activating specific heterotrimeric G proteins at the cell surface, to one of an increasing complex receptor signaling system. However, our understanding of how dimerization and oligomerization, particularly homomerization, generates functional diversity in GPCR signaling is poorly understood. For the Class A/rhodopsin subfamily of glycoprotein hormone receptors (GpHRs), di/oligomerization has been demonstrated to play a significant role in regulating its signal activity at a cellular and physiological level and even pathophysiologically. Here we will describe and discuss the developments in our understanding of GPCR oligomerization, primarily the role of homomeric receptor complexes, in both health and disease, from the study of this unique and complex subfamily of GPCRs.
2017
- Dissecting intrinsic and ligand-induced structural communication in the β3 headpiece of integrins
[Articolo su rivista]
Felline, Angelo Nicola; Ghitti, Michela; Musco, Giovanna; Fanelli, Francesca
abstract
Background: Graph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes. Methods: Protein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the β3 domains from integrins αVβ3 and αIIbβ3 in their apo and ligand-bound states. Results: Closed and open states of the β headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/β interfaces in the βI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the β1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states. Conclusions: Allosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αβα-sandwich architecture of integrin βI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the βI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process. General significance: The study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands.
2017
- Epilepsy and intellectual disability linked protein Shrm4 interaction with GABA B Rs shapes inhibitory neurotransmission
[Articolo su rivista]
Zapata, Jonathan; Moretto, Edoardo; Hannan, Saad; Murru, Luca; Longatti, Anna; Mazza, Davide; Benedetti, Lorena; Fossati, Matteo; Heise, Christopher; Ponzoni, Luisa; Valnegri, Pamela; Braida, Daniela; Sala, Mariaelvina; Francolini, Maura; Hildebrand, Jeffrey; Kalscheuer, Vera; Fanelli, Francesca; Sala, Carlo; Bettler, Bernhard; Bassani, Silvia; Smart, Trevor G.; Passafaro, Maria
abstract
Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABA B receptors (GABA B Rs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABA B R activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABA B Rs and extrasynaptic Î-subunit-containing GABA A Rs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABA B R-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.
2017
- Structural Determinants of Constitutive Activation of Gα Proteins: Transducin as a Paradigm
[Articolo su rivista]
Felline, Angelo Nicola; Mariani, Simona; Raimondi, Francesco; Bellucci, Luca; Fanelli, Francesca
abstract
Heterotrimeric guanine nucleotide-binding proteins (Gα proteins) are intracellular nanomachines deputed to signal transduction. The switch-on process requires the release of bound GDP from a site at the interface between GTPase and helical domains. Nucleotide release is catalyzed by G protein Coupled Receptors (GPCRs). Here we investigate the functional dynamics of wild type (WT) and six constitutively active mutants (CAMs) of the Gα protein transducin (Gt) by combining atomistic molecular dynamics (MD) simulations with Maxwell-Demod discrete MD (MDdMD) simulations of the receptor-catalyzed transition between GDP-bound and nucleotide-free states. Compared to the WT, Gt CAMs increase the overall fluctuations of nucleotide and its binding site. This is accompanied by weakening of native links involving GDP, α1, the G boxes, β1-β3, and α5. Collectively, constitutive activation by the considered mutants seems to associate with weakening of the interfaces between α5 and the surrounding portions and the interface between GTPase (G) and helical (H) domains. These mutational effects associate with increases in the overall fluctuations of the G and H domains, which reflect on the collective motions of the protein. Gt CAMs, with prominence to G56P, T325A, and F332A, prioritize collective motions of the H domain overlapping with the collective motions associated with receptor-catalyzed nucleotide release. In spite of different local perturbations, the mechanisms of nucleotide exchange catalyzed by activating mutations and by receptor are expected to employ similar molecular switches in the nucleotide binding site and to share the detachment of the H domain from the G domain.
2017
- Uncovering GPCR and G Protein Function by Protein Structure Network Analysis
[Capitolo/Saggio]
Fanelli, Francesca; Felline, Angelo Nicola
abstract
Protein structure network (PSN) analysis is one of the graph theory-based approaches currently used for investigating structural communication in biomolecular systems. Information on the system's dynamics can be provided by atomistic molecular dynamics (MD) simulations or coarse grained elastic network models paired with normal mode analysis (ENM-NMA). This chapter reports on selected applications of PSN analysis to uncover the structural communication in G protein coupled receptors (GPCRs) and G proteins. Strategies to highlight changes in structural communication caused by mutations, ligand and protein binding are described. Conserved amino acids, sites of misfolding mutations, or ligands acting as functional switches tend to behave as hubs in the native structure networks. Densely linked regions in the protein structure graphs could be identified as playing central roles in protein stability and function. Changes in the communication pathway fingerprints depending on the bound ligand or following amino acid mutation could be highlighted as well. A bridge between misfolding and misrouting could be established in rhodopsin mutants linked to inherited blindness. The analysis of native network perturbations by misfolding mutations served to infer key structural elements of protein responsiveness to small chaperones with implications for drug discovery.
2016
- Modulation of thermal noise and spectral sensitivity in Lake Baikal cottoid fish rhodopsins
[Articolo su rivista]
Luk, Hoi Ling; Bhattacharyya, Nihar; Montisci, Fabio; Morrow, James M.; Melaccio, Federico; Wada, Akimori; Sheves, Mudi; Fanelli, Francesca; Chang, Belinda S. W.; Olivucci, Massimo
abstract
Lake Baikal is the deepest and one of the most ancient lakes in the world. Its unique ecology has resulted in the colonization of a diversity of depth habitats by a unique fauna that includes a group of teleost fish of the sub-order Cottoidei. This relatively recent radiation of cottoid fishes shows a gradual blue-shift in the wavelength of the absorption maximum of their visual pigments with increasing habitat depth. Here we combine homology modeling and quantum chemical calculations with experimental in vitro measurements of rhodopsins to investigate dim-light adaptation. The calculations, which were able to reproduce the trend of observed absorption maxima in both A1 and A2 rhodopsins, reveal a Barlow-type relationship between the absorption maxima and the thermal isomerization rate suggesting a link between the observed blue-shift and a thermal noise decrease. A Nakanishi point-charge analysis of the electrostatic effects of non-conserved and conserved amino acid residues surrounding the rhodopsin chromophore identified both close and distant sites affecting simultaneously spectral tuning and visual sensitivity. We propose that natural variation at these sites modulate both the thermal noise and spectral shifting in Baikal cottoid visual pigments resulting in adaptations that enable vision in deep water light environments.
2016
- Small-Molecule Protein-Protein Interaction Inhibitor of Oncogenic Rho Signaling
[Articolo su rivista]
Diviani, Dario; Raimondi, Francesco; Del Vescovo, Cosmo D.; Dreyer, Elisa; Reggi, Erica; Osman, Halima; Ruggieri, Lucia; Gonano, Cynthia; Cavin, Sabrina; Box, Clare L.; Lenoir, Marc; Overduin, Michael; Bellucci, Luca; Seeber, Michele; Fanelli, Francesca
abstract
Uncontrolled activation of Rho signaling by RhoGEFs, in particular AKAP13 (Lbc) and its close homologs, is implicated in a number of human tumors with poor prognosis and resistance to therapy. Structure predictions and alanine scanning mutagenesis of Lbc identified a circumscribed hot region for RhoA recognition and activation. Virtual screening targeting that region led to the discovery of an inhibitor of Lbc-RhoA interaction inside cells. By interacting with the DH domain, the compound inhibits the catalytic activity of Lbc, halts cellular responses to activation of oncogenic Lbc pathways, and reverses a number of prostate cancer cell phenotypes such as proliferation, migration, and invasiveness. This study provides insights into the structural determinants of Lbc-RhoA recognition. This is a successful example of structure-based discovery of a small protein-protein interaction inhibitor able to halt oncogenic Rho signaling in cancer cells with therapeutic implications.
2016
- Structure network analysis to gain insights into GPCR function
[Articolo su rivista]
Fanelli, Francesca; Felline, Angelo Nicola; Raimondi, Francesco; Seeber, Michele
abstract
G protein coupled receptors (GPCRs) are allosteric proteins whose functioning fundamentals are the communication between the two poles of the helix bundle. Protein structure network (PSN) analysis is one of the graph theory-based approaches currently used to investigate the structural communication in biomolecular systems. Information on system's dynamics can be provided by atomistic molecular dynamics (MD) simulations or coarse grained elastic network models paired with normal mode analysis (ENM-NMA). The present review article describes the application of PSN analysis to uncover the structural communication in G protein coupled receptors (GPCRs). Strategies to highlight changes in structural communication upon misfolding, dimerization and activation are described. Focus is put on the ENM-NMA-based strategy applied to the crystallographic structures of rhodopsin in its inactive (dark) and signalling active (meta II (MII)) states, highlighting changes in structure network and centrality of the retinal chromophore in differentiating the inactive and active states of the receptor.
2015
- Catching Functional Modes and Structural Communication in Dbl Family Rho Guanine Nucleotide Exchange Factors
[Articolo su rivista]
Raimondi, Francesco; Felline, Angelo Nicola; Fanelli, Francesca
abstract
Computational approaches such as Principal Component Analysis (PCA) and Elastic Network Model-Normal Mode Analysis (ENM-NMA) are proving to be of great value in investigating relevant biological problems linked to slow motions with no demand in computer power. In this study, these approaches have been coupled to the graph theory-based Protein Structure Network (PSN) analysis to dissect functional dynamics and structural communication in the Dbl family of Rho Guanine Nucleotide Exchange Factors (RhoGEFs). They are multidomain proteins whose common structural feature is a DH-PH tandem domain deputed to the GEF activity that makes them play a central role in cell and cancer biology. While their common GEF action is accomplished by the DH domain, their regulatory mechanisms are highly variegate and depend on the PH and the additional domains as well as on interacting proteins. Major evolutionary-driven deformations as inferred from PCA concern the alpha6 helix of DH that dictates the orientation of the PH domain. Such deformations seem to depend on the mechanisms adopted by the GEF to prevent Rho binding, i.e. functional specialization linked to autoinhibition. In line with PCA, ENM-NMA indicates alpha6 and the linked PH domain as the portions of the tandem domain holding almost the totality of intrinsic and functional dynamics, with the alpha6/beta1 junction acting as a hinge point for the collective motions of PH. In contrast, the DH domain holds a static scaffolding and hub behavior, with structural communication playing a central role in the regulatory actions by other domains/proteins. Possible allosteric communication pathways involving essentially DH were indeed found in those RhoGEFs acting as effectors of small or heterotrimeric RasGTPases. The employed methodology is suitable for deciphering structure/dynamics relationships in large sets of homologous or analogous proteins.
2015
- Classification of Rhodopsin mutations by integrated in silico and in vitro analyses for screening of chaperon molecules to rescue misfolding.
[Abstract in Rivista]
Marigo, Valeria; Behnen, PETRA JOHANNA; Felline, Angelo Nicola; Fanelli, Francesca
abstract
Purpose: About 140 point mutations were identified in the rhodopsin gene (RHO) as cause of Autosomal Dominant Retinitis Pigmentosa (ADRP), a genetic degenerative disease causing blindness in later life. A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Characterization of the structural and molecular features of such mutants will support the development of rational drug design.
Methods: Wild type RHO and 33 different RHO mutations were analyzed in silico either in the rhodopsin form bound to retinal or in the opsin form by thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. In parallel, the same mutants were cloned in expression vectors and in vitro expressed in COS-7 cells either in the absence or presence of 9-cis retinal in the culture medium. The subcellular localization was analyzed with two monoclonal antibodies recognizing either the extracellular N-terminal or the intracellular C-terminal of RHO. Retention in the endoplasmic reticulum (ER) was assessed by analysis of co-localization with calnexin and calculation of the Pearson Correlation Coefficient (PCC) of co-localization.
Results: In silico studies revealed that the selected ADRP RHO mutations share marked abilities to impair highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates to the stability of the protein. We defined computational indices whose combination led to a structural classification of the mutants. The in vitro level of analysis revealed increased ER retention and reduction of plasma membrane localization of most of the mutants compared to wild type RHO. We found a strong correlation of the perturbation indexes calculated by in silico analyses with PCC calculated by in vitro analyses. We could also characterize different abilities of the mutated proteins to be affected by treatment with 9-cis retinal.
Conclusions: These two levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. We also developed a PCC index to evaluate the effect of retinal on protein folding and protein localization at the plasma membrane. This knowledge will be our starting point for in silico screening of compounds able to bind the retinal site and act as chaperones. The in vitro studies have developed a quantitative analysis to assess therapeutic effects of chaperone molecules.
2015
- Single Molecule Analysis of Functionally Asymmetric G Protein-coupled Receptor (GPCR) Oligomers Reveals Diverse Spatial and Structural Assemblies
[Articolo su rivista]
Jonas, Kim C.; Fanelli, Francesca; Huhtaniemi, Ilpo T.; Hanyaloglu, Aylin C.
abstract
Formation of G protein-coupled receptors (GPCRs) in to dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. We present a super-resolution imaging approach, resolving single GPCR molecules to 8nm resolution in functional asymmetric dimers and oligomers using dual-color photoactivatable dyes and localization microscopy (PD-PALM). PD-PALM of two functionally defined mutant luteinizing hormone receptors (LHRs), a ligand-binding deficient receptor (LHRB-) and a signaling deficient (LHRS-) receptor, which only function via intermolecular cooperation, favored oligomeric over dimeric formation. PD-PALM imaging of trimers and tetramers revealed specific spatial organizations of individual protomers in complexes where the ratiometric composition of LHRB- to LHRS- modulated ligand-induced signal sensitivity. Structural modeling of asymmetric LHR oligomers strongly aligned with PD-PALM imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Our findings reveal that diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.
2015
- The intellectual disability protein RAB39B selectively regulates GluA2 trafficking to determine synaptic AMPAR composition
[Articolo su rivista]
Mignogna, Maria Lidia; Giannandrea, Maila; Gurgone, Antonia; Fanelli, Francesca; Raimondi, Francesco; Mapelli, Lisa; Bassani, Silvia; Fang, Huaqiang; Van Anken, Eelco; Alessio, Massimo; Passafaro, Maria; Gatti, Silvia; Esteban, José A; Huganir, Richard; D'Adamo, Patrizia
abstract
RAB39B is a member of the RAB family of small GTPases that controls intracellular vesicular trafficking in a compartment-specific manner. Mutations in the RAB39B gene cause intellectual disability comorbid with autism spectrum disorder and epilepsy, but the impact of RAB39B loss of function on synaptic activity is largely unexplained. Here we show that protein interacting with C-kinase 1 (PICK1) is a downstream effector of GTP-bound RAB39B and that RAB39B-PICK1 controls trafficking from the endoplasmic reticulum to the Golgi and, hence, surface expression of GluA2, a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). The role of AMPARs in synaptic transmission varies depending on the combination of subunits (GluA1, GluA2 and GluA3) they incorporate. RAB39B downregulation in mouse hippocampal neurons skews AMPAR composition towards non GluA2-containing Ca(2+)-permeable forms and thereby alters synaptic activity, specifically in hippocampal neurons. We posit that the resulting alteration in synaptic function underlies cognitive dysfunction in RAB39B-related disorders.
2015
- WebPSN: a web server for high-throughput investigation of structural communication in biomacromolecules
[Articolo su rivista]
Seeber, Michele; Felline, Angelo Nicola; Raimondi, Francesco; Mariani, Simona; Fanelli, Francesca
abstract
SUMMARY: We developed a mixed Protein Structure Network (PSN) and Elastic Network Model-Normal Mode Analysis (ENM-NMA)-based strategy (i.e. PSN-ENM) to investigate structural communication in biomacromolecules. The approach starts from a Protein Structure Graph and searches for all shortest communication pathways between user-specified residues. The graph is computed on a single preferably high-resolution structure. Information on system's dynamics is supplied by ENM-NMA. The PSN-ENM methodology is made of multiple steps both in the setup and analysis stages, which may discourage inexperienced users. To facilitate its usage, we implemented WebPSN, a freely available web server that allows the user to easily setup the calculation, perform post-processing analyses and both visualize and download numerical and 3D representations of the output. Speed and accuracy make this server suitable to investigate structural communication, including allosterism, in large sets of bio-macromolecular systems. Availability and implementation: The WebPSN server is freely available at http://webpsn.hpc.unimore.it. CONTACT: fanelli@unimo.it Supplementary information: Supplementary data are available at Bioinformatics online.
2014
- Comparison of the isomerization mechanisms of human melanopsin and invertebrate and vertebrate rhodopsins.
[Articolo su rivista]
S., Rinaldi; F., Melaccio; S., Gozem; Fanelli, Francesca; M., Olivucci
abstract
Comparative modeling and ab initio multiconfigurational quantum chemistry are combined to investigate the reactivity of the human nonvisual photoreceptor melanopsin. It is found that both the thermal and photochemical isomerization of the melanopsin 11-cis retinal chromophore occur via a space-saving mechanism involving the unidirectional, counterclockwise twisting of the =C11H-C12H= moiety with respect to its Lys340-linked frame as proposed by Warshel for visual pigments [Warshel A (1976) Nature 260(5553):679-683]. A comparison with the mechanisms documented for vertebrate (bovine) and invertebrate (squid) visual photoreceptors shows that such a mechanism is not affected by the diversity of the three chromophore cavities. Despite such invariance, trajectory computations indicate that although all receptors display less than 100 fs excited state dynamics, human melanopsin decays from the excited state ∼40 fs earlier than bovine rhodopsin. Some diversity is also found in the energy barriers controlling thermal isomerization. Human melanopsin features the highest computed barrier which appears to be ∼2.5 kcal mol(-1) higher than that of bovine rhodopsin. When assuming the validity of both the reaction speed/quantum yield correlation discussed by Warshel, Mathies and coworkers [Weiss RM, Warshel A (1979) J Am Chem Soc 101:6131-6133; Schoenlein RW, Peteanu LA, Mathies RA, Shank CV (1991) Science 254(5030):412-415] and of a relationship between thermal isomerization rate and thermal activation of the photocycle, melanopsin turns out to be a highly sensitive pigment consistent with the low number of melanopsin-containing cells found in the retina and with the extraretina location of melanopsin in nonmammalian vertebrates.
2014
- Multiscale quantum chemical approaches to QSAR modeling and drug design
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
The comparative use of classical, quantum chemical (QC) ligand-based (LB) and structure-based (SB) quantitative structure-activity relationship (QSAR) results in a detailed and mechanistic-causative description, at different scales (multiscale: classical=macroscopic, LB and SB=electronic-atomistic-nanoscale) and resolution levels, of the energetics and thermodynamics of the binding event for a congeneric set of ligands and/or drugs. QC interaction propensity (reactivity) descriptors in LB QSARs provide an implicitly more accurate estimation of the enthalpic contribution to ligand-target interactions compared with classical QSAR. As for QSAR models from ab initio SB fragment molecular orbital calculations, an explicit enthalpic description of the different additive terms in the computed binding energy is obtainable. Moreover, it is possible to estimate the difference in the free energy change of the ligand-target complex formation and evaluate, on a correlative basis, the contribution of each additive free energy term to the total value.
2013
- A Mixed Protein Structure Network and Elastic Network Model
Approach to Predict the Structural Communication in Biomolecular
Systems: The PDZ2 Domain from Tyrosine Phosphatase 1E As a Case
Study
[Articolo su rivista]
Raimondi, Francesco; Felline, Angelo Nicola; M., Seeber; Mariani, Simona; Fanelli, Francesca
abstract
Graph theory is being increasingly used to study the structural
communication in biomolecular systems. This requires incorporating information on the system’s dynamics, which is time-consuming and not suitable for high-throughput
investigations. We propose a mixed Protein Structure Network (PSN) and Elastic Network Model (ENM)-based strategy, i.e., PSN-ENM, for fast investigation of allosterism in
biological systems. PSN analysis and ENM-Normal Mode Analysis (ENM-NMA) are implemented in the structural analysis software Wordom, freely available at http://wordom.
sourceforge.net/. The method performs a systematic search of the shortest communication pathways that traverse a protein structure. A number of strategies to compare the structure
networks of a protein in different functional states and to get a global picture of communication pathways are presented as well. The approach was validated on the PDZ2 domain from tyrosine phosphatase 1E (PTP1E) in its free (APO) and peptide-bound states. PDZ domains are, indeed, the systems whose structural communication and allosteric features are best characterized both in vitro and in silico. The agreement between predictions by the PSN-ENM method and in vitro evidence is remarkable and comparable to or higher than that reached by more time-consuming computational approaches tested on the same biological system. Finally, the PSN-ENM method was able to reproduce the salient communication features of unbound and bound PTP1E inferred from molecular dynamics simulations. High speed makes this method suitable for high throughput investigation of the communication pathways in large sets of biomolecular systems in different functional states.
2013
- Composti e composizioni per l'uso nella inibizione dell' interazione lbc-rhoa
[Brevetto]
Del Vescovo, Damiano; Diviani, Dario; Fanelli, Francesca; Raimondi, Francesco
abstract
The present invention relates to compounds of formula (III) and/or salts and/or derivatives thereof, or compounds of formula (IV) and/or salts and/or derivatives thereof for use in the inhibition of the Lbc-RhoA interaction and to the pharmaceutical compositions comprising the same.
The present invention relates also to compounds of formula (III) and/or salts and/or derivatives thereof, or compounds of formula (IV) and/or salts and/or derivatives thereof for use in the treatment of cancer and to the pharmaceutical compositions comprising the same.
2013
- Integrated in silico and in vitro characterization of Rhodopsin mutations and molecular mechanisms activated in photoreceptor cell death
[Poster]
Marigo, Valeria; Behnen, PETRA JOHANNA; Comitato, Antonella; DI SALVO, MARIA TERESA; Felline, Angelo Nicola; Fanelli, Francesca
abstract
Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Despite the high genetic heterogeneity of RP, ~140 point mutations were identified in the rhodopsin gene (RHO) as cause of the Autosomal Dominant form of the disease (ADRP). A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Yet, the structural and molecular features of such mutants and the cell death pathways activated by mutant RHO are obscure, hampering rational drug design.
Methods: In silico experiments on wild type RHO and 36 different mutations consisted in thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. Subcellular localization and effects of retinal as chaperone in the same mutants were characterized in vitro in COS-7 cells. Molecular death pathways activated by RHO mutation were studied in vivo in transgenic and knock-in mice bearing the P23H mutation.
Results: In silico studies revealed that ADRP RHO mutations share marked abilities to impair selected highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates in the stability core of the protein. The in vitro level of analysis revealed reduction in expression levels and plasma membrane localization of some of the mutants compared to wild type RHO as well as different abilities of the mutated proteins to be affected by 9-cis retinal. We characterized ER-stress pathways and calpain pathway activation in P23H mutant retinas. We defined the different contributions of these pathways by in vivo treatments with specific drugs blocking either ER-stress or calpains.
Conclusions: The in silico and in vitro levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. We also characterized cell death pathways activated by RHO mutants. This knowledge will be our starting point for an in silico screen of chaperone molecules to be tested in vivo.
2013
- Integrated in silico and in vitro characterization of Rhodopsin mutations causing RP4
[Abstract in Rivista]
Marigo, Valeria; Behnen, PETRA JOHANNA; Felline, Angelo Nicola; Fanelli, Francesca
abstract
Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Despite the high genetic heterogeneity of RP, ~140 point mutations were discovered in the rhodopsin gene (RHO). RHO belongs to the G protein Coupled Receptor superfamily of seven-transmembrane proteins. The vast majority of the rhodopsin mutations cause the Autosomal Dominant form (ADRP) of the disease. A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Yet, the structural and molecular features of such mutants are obscure, which hampers rational drug design.
Methods: In silico experiments on wild type RHO and 36 different mutations consisted in thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. In parallel, the same mutants were cloned in expression vectors and in vitro expressed in COS-7 cells. The subcellular localization was analyzed with two monoclonal antibodies recognizing either the extracellular N-terminal or the intracellular C-terminal of RHO. In order to define levels of expression and differences in post-translational modifications of the mutants compared to the wild type, the proteins were analyzed by Western blotting.
Results: In silico studies revealed that ADRP RHO mutations share marked abilities to impair selected highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates in the stability core of the protein. We could define a number of computational indices whose combination led to a structural classification of the mutants. The in vitro level of analysis revealed reduction in expression levels and plasma membrane localization of some of the mutants compared to wild type RHO. We also defined different abilities of the mutated proteins to be affected by 9-cis retinal.
Conclusions: These two levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. This knowledge will be our starting point for the choice of a number of mutations to be used to reveal therapeutic effect of chaperone molecules.
2013
- Light on the structural communication in Ras GTPases
[Articolo su rivista]
F., Raimondi; Felline, Angelo Nicola; G., Portella; M., Orozco; Fanelli, Francesca
abstract
The graph theory was combined with fluctuation dynamics to investigate the structural communication in four small G proteins, Arf1, H-Ras, RhoA, and Sec4.The topology of small GTPases is such that it requires the presence of the nucleotide to acquire a persistent structural network. The majority of communication paths involves the nucleotide and does not exist in the unbound forms. The latter are almost devoid of high-frequency paths. Thus, small Ras GTPases acquire the ability to transfer signals in the presence of nucleotide, suggesting that it modifies the intrinsic dynamics of the protein through the establishment of regions of hyperlinked nodes with high occurrence of correlated motions. The analysis of communication paths in the inactive (SGDP) and active (SGTP) states of the four G proteins strengthened the separation of the Ras-like domain into two dynamically distinct lobes, i.e. lobes 1 and 2, representing, respectively, the N-terminal and C-terminal halves of the domain. In the framework of this separation, inter-functional states and inter-family differences could be inferred. The structure network undergoes a reshaping depending on the bound nucleotide. Nucleotide-dependent divergences in structural communication reach the maximum in Arf1 and the minimum in RhoA. In Arf1, the nucleotide-dependent paths essentially express a communication between the G box 4 (G4) and distal portions of lobe 1. In the SGDP state the G4 communicates with the N-term, while in the SGTP state the G4 communicates with the switch II. Clear differences could be also found between Arf1 and the other three G proteins. In Arf1 the nucleotide tends to communicate with distal portions of lobe 1, whereas in H-Ras, RhoA and Sec4 it tends to communicate with a cluster of aromatic/hydrophobic amino acids in lobe 2. These differences may be linked, at least in part, to the divergent membrane anchoring modes that would involve the N-term for the Arf family and the C-term for the Rab/Ras/Rho families.
2013
- Network analysis to uncover the structural communication in GPCRs
[Articolo su rivista]
Fanelli, Francesca; Felline, Angelo Nicola; Raimondi, Francesco
abstract
Protein Structure Network (PSN) analysis is one of the graph theory-based approaches currently used to investigate the structural communication in biomolecular systems. Information on system’s dynamics can be provided by atomistic Molecular Dynamics (MD) simulations or coarse grained Elastic Network Models paired with Normal Mode Analysis (ENM-NMA).
This article describes the application of PSN analysis to uncover the structural communication in G protein Coupled Receptors (GPCRs). Strategies to highlight changes in structural communication upon misfolding mutations, dimerization, and activation are described.
Focus is put on the ENM-NMA-based strategy applied to the crystallographic structures of rhodopsin in its inactive (dark) and signaling active (Meta II (MII)) states, highlighting clear changes in the protein structure network and the centrality of the retinal chromophore in differentiating the inactive and active states of the receptor.
2013
- Network and atomistic simulations unveil the structural determinants of mutations linked to retinal diseases
[Articolo su rivista]
S., Mariani; D., Dell'Orco; Felline, Angelo Nicola; Raimondi, Francesco; Fanelli, Francesca
abstract
A number of incurable retinal diseases causing vision impairments derive from alterations in visual phototransduction. Unraveling the structural determinants of even monogenic retinal diseases would require network-centered approaches combined with atomistic simulations. The transducin G38D mutant associated with the Nougaret Congenital Night Blindness (NCNB) was thoroughly investigated by both mathematical modeling of visual phototransduction and atomistic simulations on the major targets of the mutational effect. Mathematical modeling, in line with electrophysiological recordings, indicates reduction of phosphodiesterase 6 (PDE) recognition and activation as the main determinants of the pathological phenotype. Sub-microsecond molecular dynamics (MD) simulations coupled with Functional Mode Analysis improve the resolution of information, showing that such impairment is likely due to disruption of the PDEgamma binding cavity in transducin. Protein Structure Network analyses additionally suggest that the observed slight reduction of theRGS9-catalyzed GTPase activity of transducin depends on perturbed communication between RGS9 and GTP binding site. These findings provide insights into the structural fundamentals of abnormal functioning of visual phototransduction caused by a missense mutation in one component of the signaling network. This combination of network-centered modeling with atomistic simulations represents a paradigm for future studies aimed at thoroughly deciphering the structural determinants of genetic retinal diseases. Analogous approaches are suitable to unveil the mechanism of information transfer in any signaling network either in physiological or pathological conditions.
2013
- Nucleotide binding affects intrinsic dynamics and structural communication in Ras GTPases
[Articolo su rivista]
Fanelli, Francesca; Raimondi, Francesco
abstract
The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. These proteins act biologically as molecular switches, which, cycling between OFF and ON states, play fundamental role in cell biology.
This review article summarizes the inferences from the widest computational analyses done so far on Ras GTPases aimed at providing a comprehensive structural/dynamic view of the trans-family and family-specific functioning mechanisms. The inferences from the studies derive from vast and variegated comparative analyses of the evolutionary and intrinsic flexibilities as well as of the structural communication features in the most representative G protein families in different functional states.
In spite of the low sequence similarities, the members of the Ras superfamily share the topology of the Ras-like domain, including the nucleotide binding site. GDP and GTP make very similar interactions in all GTPases and differences in their binding modes are localized around the γ-phosphate of GTP. Remarkably, such subtle local differences result in significant differences in the functional dynamics and structural communication features of the protein. In Ras GTPases, the nucleotide plays a central and active role in dictating functional dynamics, establishing the major structure network, and mediating the communication paths instrumental in function retention and specialization.
Collectively, the results of these studies support the speculation that an “extended conformational selection model” that embraces a repertoire of selection and adjustment processes is likely more suitable to describe the nucleotide behavior in these important molecular switches.
2013
- Quaternary structure predictions and structural communication features of GPCR dimers
[Capitolo/Saggio]
FANELLI, Francesca; M., Seeber; FELLINE, Angelo Nicola; CASCIARI, Daniele; RAIMONDI, Francesco
abstract
In spite of the ever increasing evidence that G protein coupled receptors (GPCR) form dimers/oligomers, the biological role(s) and structural architecture of homologous and heterologous receptor aggregation is/are, however, far from being clarified.
This chapter reviews the insights gained so far, at multiscale levels of resolution, on GPCR dimerization/oligomerization from in vitro experiments, structure predictions, and structure determinations. Focus is put on the achievement by the FiPD-based approach, which proved effective in predicting the supramolecular organization of membrane proteins including GPCRs. The combination of FiPD-based quaternary structure predictions with molecular simulations and analyses can be a valuable tool to infer the effects of dimerization on the structural communication features of a receptor dimer/oligomer bound to functionally different ligands.
Ultimately, the integration between atomistic and mesoscopic simulations is expected to be a promising tool to unveil functioning mechanisms that involve intricate protein networks.
2013
- The catalytic site structural gate of adenosine deaminase allosterically modulates ligand binding to adenosine receptors
[Articolo su rivista]
E., Gracia; D., Farré; A., Cortés; C., Ferrer Costa; M., Orozco; J., Mallol; C., Lluís; E. I., Canela; P. J., Mccormick; R., Franco; Fanelli, Francesca; V., Casadó
abstract
The enzyme adenosine deaminase (ADA) is a multifunctional protein that can both degrade adenosine and
binds extracellularly to adenosine receptors, acting as an allosteric modulator regulating the hormonal effects of
adenosine. The molecular regions of ADA responsible for the latter are unknown. In this paper, alanine
scanning mutagenesis of various ADA amino acid stretches, selected through in silico docking experiments,
allowed to identify regions of the enzyme responsible for modulating both its catalytic activity and its ability to
modulate agonist binding to A1 and A2A adenosine receptors (A1R and A2AR). The combination of
computational and in vitro experiments show that the structural gate to the catalytic site, i.e the α-1 helix
containing residues L58-I72 and the loop containing residues A184-I188 of ADA were important to maintain
both the catalytic efficiency of the enzyme and its action as an allosteric modulator of the adenosine receptors.
These data are consistent with a predicted supramolecular assembly, in which ADA bridges A2AR and CD26
and are in line with the notion that the interaction of ADA with adenosine receptors has an important role in the
immunosynapse. We propose that it is the ADA open form, but not the closed one, that is responsible for the
functional interaction with A1R and A2AR.
2012
- Modeling the structural communication in supramolecular complexes involving GPCRs
[Capitolo/Saggio]
Fanelli, Francesca
abstract
This article describes a computational strategy aimed at studying the structural communication in G-Protein Coupled Receptors (GPCRs) and G proteins. The strategy relies on comparative Molecular Dynamics (MD) simulations and analyses of wild-type (i.e., reference state) vs. mutated (i.e., perturbed state), or free (i.e., reference state) vs. bound (i.e., perturbed state) forms of a GPCR or a G protein. Bound forms of a GPCR include complexes with small ligands and/or receptor dimers/oligomers, whereas bound forms of heterotrimeric GDP-bound G proteins concern the complex with a GPCR. The computational strategy includes structure prediction of a receptor monomer (in the absence of high-resolution structure), a receptor dimer/oligomer, and a receptor-G protein complex, which constitute the inputs of MD simulations. Finally, the analyses of the MD trajectories are instrumental in inferring the structural/dynamics differences between reference and perturbed states of a GPCR or a G protein. In this respect, focus will be put on the analysis of protein structure networks and communication paths.
2012
- The KDEL receptor couples to G alpha(q/11) to activate Src kinases and regulate transport through the Golgi
[Articolo su rivista]
M., Giannotta; C., Ruggiero; M., Grossi; J., Cancino; M., Capitani; T., Pulvirenti; G. M., Consoli; C., Geraci; Fanelli, Francesca; A., Luini; M., Sallese
abstract
Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra-Golgi trafficking and is initiated by the KDEL receptor (KDEL-R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL-R signalling mechanism. We show that the KDEL-R is predicted to fold like a G-protein-coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G-protein Galpha(q/11) which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR-like mode of action of the KDEL-R and shed light on a core molecular control mechanism of intra-Golgi traffic.
2011
- Conserved amino acids participate in the structure networks deputed to intramolecular communication in the lutropin receptor
[Articolo su rivista]
K., Angelova; Felline, Angelo Nicola; M., Lee; M., Patel; D., Puett; Fanelli, Francesca
abstract
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) particularly susceptible to spontaneous pathogenic gain-of-function mutations. Protein structure network (PSN) analysis on wild-type LHR and two constitutively active mutants, combined with in vitro mutational analysis, served to identify key amino acids that are part of the regulatory network responsible for propagating communication between the extracellular and intracellular poles of the receptor. Highly conserved amino acids in the rhodopsin family GPCRs participate in the protein structural stability as network hubs in both the inactive and active states. Moreover, they behave as the most recurrent nodes in the communication paths between the extracellular and intracellular sides in both functional states with emphasis on the active one. In this respect, non-conservative loss-of-function mutations of these amino acids is expected to impair the most relevant way of communication between activating mutation sites or hormone-binding domain and G protein recognition regions.
2011
- Dimerization and ligand binding affect the structure network of A(2A) adenosine receptor
[Articolo su rivista]
Fanelli, Francesca; Felline, Angelo Nicola
abstract
G protein Coupled Receptors (GPCRs) are allosteric proteins whose functioning fundamentals are the communication between the two poles of the helix bundle. The representation of GPCR structures as networks of interacting amino acids can be a meaningful way to decipher the impact of ligand and of dimerization/oligomerization on the molecular communication intrinsic to the protein fold. In this study, we predicted likely homodimer architectures of the A(2A)R and investigated the effects of dimerization on the structure network and the communication paths of the monomeric form. The results of this study emphasize the roles of helix 1 in A(2A)R dimerization and of highly conserved amino acids in helices 1, 2, 6 and 7 in maintaining the structure network of the A(2A)R through a persistent hub behavior as well as in the information flow between the extracellular and intracellular poles of the helix bundle. The arginine of the conserved E/DRY motif, R3.50, is not involved in the communication paths but participates in the structure network as a stable hub, being linked to both D3.49 and E6.30 like in the inactive states of rhodopsin. A(2A)R dimerization affects the communication networks intrinsic to the receptor fold in a way dependent on the dimer architecture. Certain architectures retain the most recurrent communication paths with respect to the monomeric antagonist-bound form but enhancing path numbers and frequencies, whereas some others impair ligand-mediated communication networks. Ligand binding affects the network as well. Overall, the communication network that pertains to the functional dynamics of a GPCR is expected to be influenced by ligand functionality, oligomeric order and architecture of the supramolecular assembly.
2011
- Light on the structure of thromboxane A2 receptor heterodimers
[Articolo su rivista]
Fanelli, Francesca; M., Mauri; V., Capra; F., Raimondi; F., Guzzi; M., Ambrosio; G. E., Rovati; M., Parenti
abstract
The structure-based design of a mutant form of the thromboxane A(2) prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the hetero-dimeric complexes between the TPalpha and beta isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPalpha-TPbeta hetero-dimerization serves to regulate TPalpha function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution.
2011
- Nucleotide binding switches the information flow in ras GTPases
[Articolo su rivista]
Raimondi, Francesco; G., Portella; M., Orozco; Fanelli, Francesca
abstract
The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (S(GDP) and S(GTP), respectively). For all the considered systems, the intrinsic flexibility of S(GDP) was higher than that of S(GTP), suggesting that Guanine Exchange Factor (GEF) recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Galpha proteins, especially in the inactive state. Indeed, S(GDP) of Galpha(t), is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Galpha proteins), compared to GTP.
2011
- The Extreme C-Terminal Region of G{alpha}s Differentially Couples to the Luteinizing Hormone and {beta}2-Adrenergic Receptors
[Articolo su rivista]
G., Demars; Fanelli, Francesca; D., Puett
abstract
The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein alpha-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of Galpha(s) is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and beta(2)-adrenergic receptor (beta(2)-AR), a model cell system was created for the expression and manipulation of Galpha(s) subunits in LHR(+) s49 ck cells that lack endogenous Galpha(s). On the basis of studies involving truncations, mutations, and chain extensions of Galpha(s), the CT was found to be necessary for LHR and beta(2)-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the Galpha(s) CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of Galpha(s) with residues in LHR and beta(2)-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of Galpha(s) recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of beta(2)-AR are expected to contribute significantly to differences in Galpha(s) recognition by the two receptors.
2011
- Update 1 of: Computational Modeling Approaches to Structure-Function Analysis of G Protein-Coupled Receptors
[Articolo su rivista]
Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Not available
2011
- Wordom
[Software]
Seeber, Michele; Felline, Angelo Nicola; Raimondi, Francesco; Muff, Stefanie; Friedman, Ran; Rao, Francesco; Caflisch, Amedeo; Fanelli, Francesca
abstract
Wordom is a versatile, user-friendly, and efficient program for manipulation and analysis of molecular structures
and dynamics. The following new analysis modules have been added since the publication of the original Wordom
paper in 2007: assignment of secondary structure, calculation of solvent accessible surfaces, elastic network model, motion
cross correlations, protein structure network, shortest intra-molecular and inter-molecular communication paths, kinetic
grouping analysis, and calculation of mincut-based free energy profiles. In addition, an interface with the Python scripting
language has been built and the overall performance and user accessibility enhanced. The source code ofWordom (in the
C programming language) as well as documentation for usage and further development are available as an open source
package under the GNU General Purpose License from http://wordom.sf.net.
2011
- Wordom: A user-friendly program for the analysis of molecular structures, trajectories, and free energy surfaces
[Articolo su rivista]
M., Seeber; Felline, Angelo Nicola; Raimondi, Francesco; S., Muff; R., Friedman; F., Rao; A., Caflisch; Fanelli, Francesca
abstract
Wordom is a versatile, user-friendly, and efficient program for manipulation and analysis of molecular structures and dynamics. The following new analysis modules have been added since the publication of the original Wordom paper in 2007: assignment of secondary structure, calculation of solvent accessible surfaces, elastic network model, motion cross correlations, protein structure network, shortest intra-molecular and inter-molecular communication paths, kinetic grouping analysis, and calculation of mincut-based free energy profiles. In addition, an interface with the Python scripting language has been built and the overall performance and user accessibility enhanced. The source code of Wordom (in the C programming language) as well as documentation for usage and further development are available as an open source package under the GNU General Purpose License from http://wordom.sf.net. (c) 2010 Wiley Periodicals, Inc. J Comput Chem, 2011.
2010
- Computational quantum chemistry and adaptive ligand modeling in mechanistic QSAR
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
Drugs are adaptive molecules. They realize this peculiarity by generating different ensembles of prototropic forms and conformers that depend on the environment. Among the impressive amount of available computational drug discovery technologies, quantitative structure-activity relationship approaches that rely on computational quantum chemistry descriptors are the most appropriate to model adaptive drugs. Indeed, computational quantum chemistry descriptors are able to account for the variation of the intramolecular interactions of the training compounds, which reflect their adaptive intermolecular interaction propensities. This enables the development of causative, interpretive and reasonably predictive quantitative structure-activity relationship models, and, hence, sound chemical information finalized to drug design and discovery.
2010
- Deciphering the deformation modes associated with function retention and specialization in members of the Ras superfamily
[Articolo su rivista]
Raimondi, Francesco; M., Orozco; Fanelli, Francesca
abstract
The evolutionary and physical deformability patterns of members of the Ras GTPase superfamily were investigated by Principal Component and Elastic Network-Normal Mode analyses. The study helped to decipher the dynamics information encrypted into the conserved core and to separate the trans-family intrinsic flexibility associated with a common function from the protein motions related to functional specialization of selected families or family members. The conserved core is dynamically divided into two lobes. The deformation modes, which allow the Ras GTPases to accomplish their switching function, are conserved along evolution and are localized in lobe 1 portions close to the nucleotide. These modes lead to functional specialization when associated with evolution-driven deformations of protein portions essentially located in lobe 2, distal from the nucleotide, and involved in peculiar interactions with membrane, guanine nucleotide exchange factors, or effectors. Overall, a complete picture of the functional and evolutionary dynamics of the Ras superfamily emerges.
2010
- Structural insights into retinitis pigmentosa from unfolding simulations of rhodopsin mutants
[Articolo su rivista]
Fanelli, Francesca; M., Seeber
abstract
Disease-causing missense mutations in membrane proteins, such as rhodopsin mutations associated with the autosomal dominant form of retinitis pigmentosa (ADRP), are often linked to defects in folding and/or trafficking. The mechanical unfolding of wild-type rhodopsin was compared with that of 20 selected ADRP-linked mutants more or less defective in folding and retinal binding. Rhodopsin fold is characterized by networks of amino acids in the retinal and G-protein binding sites likely to play a role in the stability and function of the protein. The distribution of highly connected nodes in the network reflects the existence of a diffuse intramolecular communication inside and between the 2 poles of the helix bundle, which makes pathogenic mutations share similar phenotypes irrespective of topological and physicochemical differences between them. Because of this communication, the ADRP-linked rhodopsin mutations share a more or less marked ability to impair selected hubs in the protein structure network. The extent of this structural effect relates to the severity of the biochemical defect caused by mutation. The investigative strategy employed in this study is likely to apply to all structurally known membrane proteins particularly susceptible to misassembly-causing mutations.
2010
- Structure-activity relationships in 1,4-benzodioxan-related compounds. 10. Novel α1-adrenoreceptor antagonists related to openphendioxan: synthesis, biological evaluation, and α1d computational study
[Articolo su rivista]
A., Carrieri; A., Piergentili; F., Del Bello; M., Giannella; M., Pigini; A., Leonardi; Fanelli, Francesca; W., Quaglia
abstract
A series of novel openphendioxan analogues were synthesized and tested at alpha(1)-adrenoreceptor (AR) subtypes by binding and functional assays. The alpha(1d)-AR binding profile was also examined by means of 2D, 3D-QSAR together with docking studies. Multiple regression analysis suggested the relevance of adequate number of heteroatoms in the whole molecule and of passive membrane diffusion to enhance alpha(1d)-AR affinity. Docking simulations against a computational structural model of the biological target further proved this evidence and furnished support for chemiometric analysis, where polar, electrostatic, hydrophobic and shape effects of the ortho substituents in the phenoxy terminal, most likely governing ligand binding, helped the depiction of pharmacophore hypothesis for the examined ligands data set.
2010
- Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants
[Articolo su rivista]
M., Ambrosio; Fanelli, Francesca; S., Brocchetti; Raimondi, Francesco; M., Mauri; G. E., Rovati; V., Capra
abstract
In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129((3.49)) or E240((6.30)) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G(q) and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM.
2010
- The luteinizing hormone receptor: insights into structure-function relationships and hormone-receptor-mediated changes in gene expression in ovarian cancer cells
[Articolo su rivista]
D., Puett; K., Angelova; M. R., da Costa; S. W., Warrenfeltz; Fanelli, Francesca
abstract
The luteinizing hormone receptor (LHR), one of the three glycoprotein hormone receptors, is necessary for critical reproductive processes, including gonadal steroidogenesis, oocyte maturation and ovulation, and male sex differentiation. Moreover, it has been postulated to contribute to certain neoplasms, particularly ovarian cancer. A member of the G protein-coupled receptor family, LHR contains a relatively large extracellular domain responsible for high affinity hormone binding; transmembrane activation then leads to G protein coupling and subsequent second messenger production. This review deals with recent advances in our understanding of LHR structure and structure-function relationships, as well as hormone-mediated changes in gene expression in ovarian cancer cells expressing LHR. Suggestions are also made for critical gaps that need to be filled as the field advances, including determination of the three-dimensional structure of inactive and active receptor, elucidation of the mechanism by which hormone binding to the extracellular domain triggers the activation of Gs, clarification of the putative roles of LHR in non-gonadal tissues, and the role, if any, of activated receptor in the development or progression of ovarian cancer.
2009
- Adenosine A(2A) Receptor-Antagonist/Dopamine D(2) Receptor-Agonist Bivalent Ligands as Pharmacological Tools to Detect A(2A)-D(2) Receptor Heteromers
[Articolo su rivista]
A., Soriano; R., Ventura; A., Molero; R., Hoen; V., Casadó; A., Cortés; Fanelli, Francesca; F., Albericio; C., Lluís; R., Franco; M., Royo
abstract
Adenosine A(2A) (A(2A)R) and dopamine D(2) (D(2)R) receptors mediate the antagonism between adenosinergic and dopaminergic transmission in striatopallidal GABAergic neurons and are pharmacological targets for the treatment of Parkinson's disease. Here, a family of heterobivalent ligands containing a D(2)R agonist and an A(2A)R antagonist linked through a spacer of variable size was designed and synthesized to study A(2A)R-D(2)R heteromers. Bivalent ligands with shorter linkers bound to D(2)R or A(2A)R with higher affinity than the corresponding monovalent controls in membranes from brain striatum and from cells coexpressing both receptors. In contrast, no differences in affinity of bivalent versus monovalent ligands were detected in experiments using membranes from cells expressing only one receptor. These findings indicate the existence of A(2A)R-D(2)R heteromers and of a simultaneous interaction of heterobivalent ligands with both receptors. The cooperative effect derived from the simultaneous interaction suggests the occurrence of A(2A)R-D(2)R heteromers in cotransfected cells and in brain striatum. The dopamine/adenosine bivalent action could constitute a novel concept in Parkinson's disease pharmacotherapy.
2009
- Computational Screening of Rhodopsin Mutations Associated with Retinitis Pigmentosa
[Articolo su rivista]
Felline, Angelo Nicola; M., Seeber; F., Rao; Fanelli, Francesca
abstract
Retinitis pigmentosa (RP) refers to a group of debilitating, hereditary disorders that cause severe visual impairment in as many as 1.5 million patients worldwide. Rhodopsin mutations account for > 25% of the autosomal dominant form of the disease (ADRP). Forty artificial and ADRP-associated mutations located in the second extracellular loop (EL2) that folds into a twisted beta-hairpin were screened through replica exchange molecular dynamics (REMD) simulations using the FACTS implicit solvent model. According to in vitro experiments, ADRP-linked mutants fail to express at the plasma membrane and/or to reconstitute with 11-cis-retinal, indicative of variable defects in protein folding and/or stability. The computational protocol was first probed on the protein G C-terminal beta-hairpin, proving the effectiveness of the implicit solvent model in reproducing the free energy landscape of beta-hairpin formation. Eight out of the 40 EL2 mutants resulted in misfolding effects on the native beta-hairpin structure, consistent with in vitro evidence that they all share severe impairments in folding/expression. Five mutants displayed moderate misfolding attitudes, whereas the remaining 27 mutants, overall characterized by milder effects on rhodopsin expression, did not perturb significantly the conformational behavior of the native beta-hairpin but are expected to exert variably disturbing effects on the native interactions of the loop with the chromophore and/or the surrounding receptor domains. Collectively, the results of this study add structural insight to the poorly resolved biochemical behavior of selected class II ADRP mutations, a fundamental step toward an understanding of the atomistic causes of the disease.
2009
- Computational modeling of intramolecular and intermolecular communication in GPCRs
[Articolo su rivista]
Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe; Raimondi, Francesco; M., Seeber
abstract
Intramolecular and intermolecular communication is a privileged issue in G protein-Coupled Receptor (GPCR) function as the prominent role of these receptors is to respond to extracellular signals by catalyzing nucleotide exchange in intracellular G proteins. In the last decade or so we have applied much effort in elaborating computational strategies to infer the mechanisms of intramolecular and intermolecular communication in a number of GPCRs of the rhodopsin family. In this article, we review the most relevant achievements on the matter. In summary, the receptor sites of activating mutations or ligand-binding communicate with a common allosteric site in the cytosolic domains. This was inferred from the observation that local perturbations by activating mutations or ligands correlate with increases in solvent accessibility of the neighborhoods of the highly conserved E/DRY receptor motif. The latter turned out to be the primary recognition point for the C-terminus of the G protein alpha-subunit, independent of the receptor or the G protein type. In spite of the highly composite nature of the receptor-G protein interface, receptor contacts with the C-terminus of the alpha5-helix seem to be the major players in the receptor-catalyzed formation of a nucleotide exit route. The latter would lie in between the alphaF-helix and the beta6/alpha5 loop, which detach from each other upon receptor binding, giving solvent accessibility to the nucleotide. A worthy inference of the studies is that GPCRs employ common pathways for the transfer of functionally relevant information.
2009
- Ligand-Receptor Communication and Drug Design
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
Ligand-protein and protein-protein interactions play a pivotal role in any cellular process and function by means of complex and dynamic mechanisms that involve sophisticated intra- and intermolecular communication pathways. The deeper understanding of the molecular and structural mechanisms of these pathways of chemical information transfer constitutes the foundations of rational druggable target discovery and drug design. In this context the role of both molecular recognition/communication between the interacting partners and their quantitative/dynamic description constitute the crucial point. In this respect, many approaches at different level of complexity have been developed and applied to different druggable target like enzymes, membrane receptors and protein assembly. They mainly differ in the accuracy and resolution level of molecular description and, hence, in the derived quantitative molecular descriptors/predictors and ligand-target models. In this review, we will try to illustrate some selected examples of ligand-target receptor protein models, by comparatively considering both series of ligands (ligand-based communication modeling) and ligand-target complexes (target-based communication modeling) in order to describe the relevant structural/dynamic features of chemical information transfer in the ligand/drug design endeavour.
2009
- Network-level analysis of light adaptation in rod cells under normal and altered conditions
[Articolo su rivista]
D., Dell’Orco; H., Schmidt; S., Mariani; Fanelli, Francesca
abstract
Photoreceptor cells finely adjust their sensitivity and electrical response according to changes in light stimuli as a direct consequence of the feedback and regulation mechanisms in the phototransduction cascade. In this study, we employed a systems biology approach to develop a dynamic model of vertebrate rod phototransduction that accounts for the details of the underlying biochemistry. Following a bottom-up strategy, we first reproduced the results of a robust model developed by Hamer et al. (Vis. Neurosci., 2005, 22(4), 417), and then added a number of additional cascade reactions including: (a) explicit reactions to simulate the interaction between the activated effector and the regulator of G-protein signalling (RGS); (b) a reaction for the reformation of the G-protein from separate subunits; (c) a reaction for rhodopsin (R) reconstitution from the association of the opsin apoprotein with the 11-cis-retinal chromophore; (d) reactions for the slow activation of the cascade by opsin. The extended network structure successfully reproduced a number of experimental conditions that were inaccessible to prior models. With a single set of parameters the model was able to predict qualitative and quantitative features of rod photoresponses to light stimuli ranging over five orders of magnitude, in normal and altered conditions, including genetic manipulations of the cascade components. In particular, the model reproduced the salient dynamic features of the rod from Rpe65(-/-) animals, a well established model for Leber congenital amaurosis and vitamin A deficiency. The results of this study suggest that a systems-level approach can help to unravel the adaptation mechanisms in normal and in disease-associated conditions on a molecular basis.
2008
- An intracellular loop (IL2) residue confers different basal constitutive activities to the human lutropin receptor and human thyrotropin receptor through structural communication between IL2 and helix 6, via helix 3
[Articolo su rivista]
X., Feng; T., Muller; D., Mizrachi; Fanelli, Francesca; AND D. L., Segaloff
abstract
The human lutropin receptor (hLHR) and human TSH receptor (hTSHR) are G protein-coupled receptors that play key roles in reproductive and thyroid physiology, respectively. We show using a quantitative assessment of cAMP production as a function of cell surface receptor expression that the hTSHR possesses greater basal constitutive activity than the hLHR. Further studies were undertaken to test the hypothesis that different potential Gs-coupling motifs identified in IL2 of the hTSHR and hLHR contribute to their different basal constitutive activities. Although mutating the receptors to interchange their potential Gs-coupling motifs reversed their relative activities, we show this to be due to the swapping of one IL2 residue (Q476 in the hLHR; R531 in the hTSHR). Molecular dynamics simulations show that the effect of the hLHR(Q476R) mutation, switching the structural features of the hLHR toward those of the hTSHR, is greater than the switching effect of the hTSHR(R531Q) mutant toward the hLHR. The structural model of the hLHR(Q476R) mutant can be considered as a hybrid of wild-type (wt) hTSHR and constitutively active mutant hLHR forms. In this hLHR(Q476R) mutant, IL2 adopts a structure similar to IL2 of the wt hTSHR, but it shares with the hLHR constitutively active mutant the solvent exposure and the reciprocal arrangement of helices 3, 5, and 6, including the weakening of the wt native R3.50-D6.30 interaction. Our results suggest a H3-mediated structural connection between IL2 and the cytosolic extension of H6. Thus, IL2 contributes significantly to the inactive and active state ensembles of these G protein-coupled receptors.
2008
- Computational modeling of selective pharmacophores at the alpha1-adrenergic receptors
[Capitolo/Saggio]
Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
1. Introduction2. Ligand-based and receptor-based pharmacophore modeling and QSAR analysis3. The general 1-AR pharmacophore3.1. Ligand-based pharmacophore and virtual screening 3.1.1. Prazosin analogues (2,4-diamino-6,7-dimethoxyquinazoline derivatives)3.1.2. 1,4-benzodioxan (WB-4101) related compounds3.1.3. Arylpiperazine derivatives4. Modeling the 1-AR subtype selectivities of different antagonist classes4.1.1. Supermolecule based subtype pharmacophore and QSAR models4.1.2. Ligand based subtype pharmacophores4.1.3. Receptor-based subtype pharmacophore and ligand-target/antitarget interaction-based QSAR5. Antitarget modeling of biogenic amine-binding GPCRs 6. Inverse agonism: an alternative way to interpret the 1a/1b-selectivity issue6.1 In vitro functional screening of 1a and 1b ligands.6.2 Ligand- and receptor-based structural interpretation of inverse agonism67. Concluding remarks 67.1. From molecules to pharmacophores to descriptors to models 78. Perspectives8.1. Multiscale computational modeling of GPCRs8.2. Molecular systems biology and pharmacology: “network drugs”
2008
- Contributions of Intracellular Loops 2 and 3 of the Lutropin Receptor in Gs Coupling
[Articolo su rivista]
K., Angelova; Fanelli, Francesca; D., Puett
abstract
A number of amino acids essential for Gs coupling, i.e. hot spots, were identified after in vitro Ala-scanning mutagenesis of the cytosolic extensions of helices 3, 5, and 6 and of intracellular loops 2 and 3 (IL2 and IL3) of the human LH receptor (LHR). Consistent with the results of in vitro experiments involving ligand binding and ligand-mediated signaling in transiently transfected human embryonic kidney 293 cells, computational modeling of the isolated receptor and of the receptor-G protein complexes suggests an important role of the cytosolic extension of helix 3 and the N-terminal portion of the IL2 in Gs(alpha) interaction, whereas the contribution of IL3 is marginal. Mapping the hot spots into the computational models of LHR and the LHR-Gs complexes allowed for a distinction between receptor sites required for intramolecular structural changes (i.e. I460, T461, H466, and I549) and receptor sites more likely involved in G protein recognition (i.e. R464, T467, I468, Y470, Y550, and D564). The latter sites include the highly conserved arginine of the (E/D)R(Y/W) motif, which is therefore likely to be a receptor recognition point for Gs rather than a switch of receptor activation. The results of in vitro and in silico experiments carried out in this study represent the first comprehensive delineation of functionality of the individual residues in the intracellular domains of LHR and establish potential switches of receptor activation as well as a map of the primary receptor recognition sites for Gs. A novel way to consider constitutively active mutants was inferred from this study, i.e. receptor states with improved complementarity for the G protein compared to the wild-type receptor.
2008
- Dark and photoactivated rhodopsin share common binding modes to transducin
[Articolo su rivista]
Fanelli, Francesca; D., Dell'Orco
abstract
The structure of the photoactivated deprotonated rhodopsin intermediate was compared with two different structures of dark rhodopsin. Structure comparisons relied on the computation of molecular indices and on docking simulations with heterotrimeric transducin (Gt). The results of this study provide the first evidence that dark and photoactivated rhodopsins share a common recognition mode to Gt, characterized by the docking of the Gt alpha C-tail in the proximity to the E/DRY motif of rhodopsin.
2008
- Homodimerization of neurotensin 1 receptor involves helices 1, 2, and 4: Insights from quaternary structure predictions and dimerization free energy estimations
[Articolo su rivista]
C., Casciari; D., Dell'Orco; Fanelli, Francesca
abstract
A computational approach based upon rigid-body docking, ad hoc filtering, and cluster analysis has been combined with a protocol for dimerization free energy estimations to predict likely interfaces in the neurotensin 1 receptor (NTS1) homodimers. The results of this study suggest that the likely intermonomer interfaces compatible with in vitro binding affinity constants essentially involve helices 1, 2, and 4 and do not include disulfide bridges. The correlative model initially developed on Glycophorin A and herein extended to a G protein-Coupled Receptor (GPCR) appears to be a useful tool for estimating the association free energies of transmembrane proteins independent of the size and shape of the interface. In the desirable future cases, in which in vitro intermonomer binding affinities will be available for other GPCRs, such a correlative model will work as an additional criterion for helping in the selection of the most likely dimers.
2008
- Mechanisms of inter- and intramolecular communication in GPCRs and G proteins
[Articolo su rivista]
Raimondi, F; Seeber, M; DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
This study represents the first attempt to couple, by computational experiments, the mechanisms of intramolecular and intermolecular communication concerning a guanidine nucleotide exchange factor (GEF), the thromboxane A2 receptor (TXA2R), and the cognate G protein (Gq) in its heterotrimeric GDP-bound state. Two-way pathways mediate the communication between the receptor-G protein interface and both the agonist binding site of the receptor and the nucleotide binding site of the G protein. The increase in solvent accessibility in the neighborhoods of the highly conserved E/DRY receptor motif, in response to agonist binding, is instrumental in favoring the penetration of the C-terminus of Gqalpha in between the cytosolic ends of H3, H5, and H6. The arginine of the E/DRY motif is predicted to be an important mediator of the intramolecular and intermolecular communication involving the TXA2R. The receptor-G protein interface is predicted to involve multiple regions from the receptor and the G protein alpha-subunit. However, receptor contacts with the C-terminus of the alpha5-helix seem to be the major players in the receptor-catalyzed motion of the alpha-helical domain with respect to the Ras-like domain and in the formation of a nucleotide exit route in between the alphaF-helix and beta6/alpha5 loop of Gqalpha. The inferences from this study are of wide interest, as they are expected to apply to the whole rhodopsin family, given also the considerable G protein promiscuity.
2008
- Quaternary structure predictions and estimation of mutational effects on the free energy of dimerization of the OMPLA protein
[Articolo su rivista]
D., Dell'Orco; D., Casciari; Fanelli, Francesca
abstract
This study represents an extension to the outer membrane phospholipase A protein (OMPLA) of the docking-based protocols previously developed for quaternary structure predictions of transmembrane oligomeric proteins and for estimating mutational effects on the thermodynamics of protein-protein and protein-DNA association. Predictions of the likely architecture of OMPLA homo-dimers were carried out on 31 different forms of the monomer, 30 of which were variants of the unbound state. In all the test cases but the ones characterized by combined deletions of the 98-110 and 145-153 segments (L2 and L3, respectively), native-like complexes could be predicted, independent of the bound or unbound state of the structural model, of side chain conformation and presence or absence of amino acid deletions at the putative inter-monomer interface. The protocol for estimating mutational effects on the thermodynamics of protein-protein association proved effective as well. In fact, it was possible to estimate correctly the effects of five mutants on the free energy of dimerization of the sulfonylated form of OMPLA. The integrity of L2 and either one of the L1, L3 and L4 loops turned out to be more important than sulfonylation for the achievement of the native dimeric architecture. On the other hand, sulfonylation seems to be essential for a favorable dimerization energetics.
2008
- Target Flexibility: An Emerging Consideration in Drug Discovery and Design
[Articolo su rivista]
Cozzini, P; Kellogg, Ge; Spyrakis, Francesca; Abraham, Dj; Costantino, G; Emerson, A; Fanelli, Francesca; Gohlke, H; Kuhn, La; Morris, Gm; Orozco, M; Pertinhez, Ta; Rizzi, M; Sotriffer, C.
abstract
As a J. Med. Chem. Perspective, this article does not have an abstract.
2007
- A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein
[Articolo su rivista]
Puett, D; Li, Y; Demars, G; Angelova, K; Fanelli, Francesca
abstract
The luteinizing hormone receptor (LHR) is one of eight members in a cluster of the rhodopsin family of the large G protein-coupled receptor (GPCR) superfamily that contains some 800-900 genes in the human genome. LHR, along with its paralogons, follicle stimulating hormone receptor (FSHR) and thyroid stimulating hormone receptor, form one of the three classes in this cluster; the two other classes contain the relaxin-binding GPCRs and orphan GPCRs. These GPCRs are characterized by a relatively large ectodomain (ECD) containing leucine-rich-repeats (LRRs); in the class of glycoprotein hormone receptors, the LRR region is capped by N-terminal and C-terminal cysteine-rich regions. Binding of human chorionic gonadotropin (hCG) or luteinizing hormone to the LHR-ECD triggers a conformational change of the transmembrane region of the receptor facilitating binding and activation of Gs, followed by effector enzyme activation and subsequent intracellular signaling. Viewing LHR as a transmembrane anchoring protein that sequentially binds hCG and Gs to give the hCG-LHR-Gs complex, numerous interactions and conformational changes must be considered. There is, unfortunately, a paucity of structural data on LHR, but crystal structures exist for hCG, the homologous FSH-FSHR-ECD (N-terminal fragment) complex, rhodopsin (in the inactive state), an active form of Galphas (transducin), and the betagamma heterodimer. Using a combined experimental (site-directed mutagenesis followed by characterization in transfected cells) and computational (homology modeling and molecular dynamics simulations) approach, good working models are being developed for the protein-protein interaction faces and, in some cases, the ensuing conformational changes induced by complex formation. hCG binding to the LHR-ECD appears to involve several LRRs; LHR activation can be described in terms of disrupting a network of H-bonds in the cytosolic halves of helices 1-3, 6, and 7; and binding of LHR to Gs involves, in large part, intracellular loop 2 binding, presumably to Gsalpha at its C-terminus. Major gaps exist in our understanding at the molecular level of the six-polypeptide chain complex, hCG-LHR-Gs, but considerable progress has been made in the past few years.
2007
- Dimerization of the lutropin receptor: Insights from computational modeling
[Articolo su rivista]
Fanelli, Francesca
abstract
A computational approach based upon rigid-body docking, ad hoc filtering, and cluster analysis has been carried out to predict likely interfaces in LHR homodimers. Quaternary structure predictions emphasize the role of helices 4, 5 and 6, with prominence to helix 4, in mediating inter-monomer interactions. Intermolecular interactions essentially involve the transmembrane domains rather than the hydrophilic loops and do not implicate disulfide bridges.Collectively, molecular dynamics simulations on the isolated receptor and computational modeling of LHR homodimerization suggest that mutation-induced LHR activation favors H4-H4 contacts involving the highly conserved W491 from both the receptors monomers.
2007
- In silico screening of mutational effects on enzyme-proteic inhibitor affinity: A docking-based approach
[Articolo su rivista]
D., Dell'Orco; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
BACKGROUND: Molecular recognition between enzymes and proteic inhibitors is crucial for normal functioning of many biological pathways. Mutations in either the enzyme or the inhibitor protein often lead to a modulation of the binding affinity with no major alterations in the 3D structure of the complex. RESULTS: In this study, a rigid body docking-based approach has been successfully probed in its ability to predict the effects of single and multiple point mutations on the binding energetics in three enzyme-proteic inhibitor systems. The only requirement of the approach is an accurate structural model of the complex between the wild type forms of the interacting proteins, with the assumption that the architecture of the mutated complexes is almost the same as that of the wild type and no major conformational changes occur upon binding. The method was applied to 23 variants of the ribonuclease inhibitor-angiogenin complex, to 15 variants of the barnase-barstar complex, and to 8 variants of the bovine pancreatic trypsin inhibitor-beta Trypsin system, leading to thermodynamic and kinetic estimates consistent with in vitro data. Furthermore, simulations with and without explicit water molecules at the protein-protein interface suggested that they should be included in the simulations only when their positions are well defined both in the wild type and in the mutants and they result to be relevant for the modulation of mutational effects on the association process. CONCLUSION: The correlative models built in this study allow for predictions of mutational effects on the thermodynamics and kinetics of association of three substantially different systems, and represent important extensions of our computational approach to cases in which it is not possible to estimate the absolute free energies. Moreover, this study is the first example in the literature of an extensive evaluation of the correlative weights of the single components of the ZDOCK score on the thermodynamics and kinetics of binding of protein mutants compared to the native state.Finally, the results of this study corroborate and extend a previously developed quantitative model for in silico predictions of absolute protein-protein binding affinities spanning a wide range of values, i.e. from -10 up to -21 kcal/mol. The computational approach is simple and fast and can be used for structure-based design of protein-protein complexes and for in silico screening of mutational effects on protein-protein recognition.
2007
- In silico screening of mutational effects on transmembrane helix dimerization: insights from rigid-body docking and molecular dynamics simulations
[Articolo su rivista]
D., Dell'Orco; DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
In this study, a docking-based protocol has been probed for its ability to predict the effects of 32 single and double mutations on glycophorin A (GpA) homodimerization. Rigid-body docking simulations have been paralleled by molecular dynamics (MD) simulations in implicit membrane. The rigid-body docking-based approach proved effective in reconstituting the native architecture of the GpA dimer for the wild type and the wild-type-like mutants. The good correlative models between the intermolecular interaction descriptors derived both by rigid-body docking simulations and by MD simulations and experimental relative free energies support the assumption that the mutation-induced changes in the association free energy of GpA helices are essentially ascribed to differences in the packing interactions, whereas almost all the variations in the entropic contributions to the association free energy are constant and/or negligible. The MD-based models achieved provide insights into the structural determinants for disruptive and restoring mutational effects. The computational approaches presented in this study are fast and effective, and constitute simple and promising tools for in silico screening of mutational effects on the association properties of integral membrane proteins.
2007
- Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations.
[Articolo su rivista]
M., Zhang; Y. X., Tao; G. L., Ryan; X., Feng; Fanelli, Francesca; D. L., Segaloff
abstract
In contrast to the human lutropin receptor (hLHR), very few naturally occurring activating mutations of the structurally related human follitropin receptor (hFSHR) have been identified. The present study was undertaken to determine if one aspect underlying this discrepancy might be a general resistance of the hFSHR to mutation-induced constitutive activity. Five different mutations were introduced into both the hLHR and hFSHR (four based on activating mutations of the hLHR gene, one based on an activating mutation of the hFSHR gene). Our results demonstrate that hFSHR constitutively activating mutants (CAMs) were not as active as hLHR CAMs containing the comparable mutation. Furthermore, although all hFSHR CAMs exhibited strong promiscuous activation by high concentrations of the other glycoprotein hormone receptors, hLHR CAMs showed little or no promiscuous activation. Our in vitro findings are consistent with in vivo observations of known pathophysiological conditions associated with hLHR CAMs, but not hFSHR CAMs, and with promiscuous activation of hFSHR CAMs, but not hLHR CAMs. Computational experiments suggest that the mechanisms through which homologous mutations increase the basal activity of the hLHR and the hFSHR are similar. This is particularly true for the strongest CAMs like L460(3.43)R. Disparate properties of the hLHR versus hFSHR CAMs may, therefore, be due to differences in shape and electrostatics features of the solvent-exposed cytosolic receptor domains involved in the receptor-G protein interface rather than to differences in the nature of local perturbation at the mutation site or in the way local perturbation is transferred to the putative G protein binding domains.
2007
- Monomeric dark rhodopsin holds the molecular determinants for transducin recognition: insights from computational analysis
[Articolo su rivista]
D., Dell'Orco; M., Seeber; Fanelli, Francesca
abstract
In this computational study, we have investigated the implications of rhodopsin (Rho) oligomerization in transducin (Gt) recognition. The results of docking simulations between heterotrimeric Gt and monomeric, dimeric and tetrameric inactive Rho corroborate the hypothesis that Rho and Gt can be found coupled already in the dark. Moreover, our extensive computational analysis suggests that the most likely Rho:Gt stoichiometry is the 1:1 one. This means that the essential molecular determinants for Gt recognition and activation are contained in one Rho monomer. In this respect, the complex between one Rho molecule and one heterotrimeric Gt should be considered as the functional unit.
2006
- Constitutive activity at the alpha1-adrenoceptors: past and future implications
[Capitolo/Saggio]
S., Cotecchia; Fanelli, Francesca; A., Scheer; T., Costa
abstract
Constitutive activity at the alpha1-adrenoceptors: past and future implications
2006
- FIPD
[Software]
Casciari, Daniele; Seeber, Michele; Fanelli, Francesca
abstract
The FIPD software serves to the postprocessing of the ZDOCK output
2006
- Inactive and active states and supramolecular organization of GPCRs: insights from computational modeling
[Articolo su rivista]
Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Herein we make an overview of the results of our computational experiments aimed at gaining insight into the molecular mechanisms of GPCR functioning either in their normal conditions or when hit by gain-of-function or loss-of-function mutations. Molecular simulations of a number of GPCRs in their wild type and mutated as well as free and ligand-bound forms were instrumental in inferring the structural features, which differentiate the mutation- and ligand-induced active from the inactive states. These features essentially reside in the interaction pattern of the E/DRY arginine and in the degree of solvent exposure of selected cytosolic domains. Indeed, the active states differ from the inactive ones in the weakening of the interactions made by the highly conserved arginine and in the increase in solvent accessibility of the cytosolic interface between helices 3 and 6. Where possible, the structural hallmarks of the active and inactive receptor states are translated into molecular descriptors useful for in silico functional screening of novel receptor mutants or ligands. Computational modeling of the supramolecular organization of GPCRs and their intracellular partners is the current challenge toward a deep understanding of their functioning mechanisms.
2006
- Prediction of MEF2A-DNA interface by rigid body docking: A tool for fast estimation of protein mutational effects on DNA binding
[Articolo su rivista]
Fanelli, Francesca; Ferrari, Stefano
abstract
The protein-protein docking algorithm ZDOCK has been challenged for the first time to predict protein-DNA contacts. The computational approach defined in this study has proven effectiveness in fast in silico estimations of mutational effects of the MEF2A transcription factor on DNA binding.
2006
- Quaternary structure predictions of transmembrane proteins starting from the monomer: a docking-based approach
[Articolo su rivista]
D., Casciari; M., Seeber; Fanelli, Francesca
abstract
BackgroundWe introduce a computational protocol for effective predictions of the supramolecular organization of integral transmembrane proteins, starting from the monomer. Despite the demonstrated constitutive and functional importance of supramolecular assemblies of transmembrane subunits or proteins, effective tools for structure predictions of such assemblies are still lacking. Our computational approach consists in rigid-body docking samplings, starting from the docking of two identical copies of a given monomer. Each docking run is followed by membrane topology filtering and cluster analysis. Prediction of the native oligomer is therefore accomplished by a number of progressive growing steps, each made of one docking run, filtering and cluster analysis. With this approach, knowledge about the oligomerization status of the protein is required neither for improving sampling nor for the filtering step. Furthermore, there are no size-limitations in the systems under study, which are not limited to the transmembrane domains but include also the water-soluble portions.ResultsBenchmarks of the approach were done on ten homo-oligomeric membrane proteins with known quaternary structure. For all these systems, predictions led to native-like quaternary structures, i.e. with Cα-RMSDs lower than 2.5 Å from the native oligomer, regardless of the resolution of the structural models.ConclusionCollectively, the results of this study emphasize the effectiveness of the prediction protocol that will be extensively challenged in quaternary structure predictions of other integral membrane proteins.
2006
- Sequential unfolding of individual helices of bacterioopsin observed in molecular dynamics simulations of extraction from the purple membrane
[Articolo su rivista]
M., Seeber; Fanelli, Francesca; E., Paci; A., Caflisch
abstract
Multiple molecular dynamics simulations of bacterioopsin pulling from its C-terminus show that its alpha-helices unfold individually. In the first metastable state observed in the simulations, helix G is unfolded at its C-terminal segment while the rest of helix G (residues 200-216) is folded and opposes resistance because of a salt-bridge network consisting of Asp-212 and Lys-216 on helix G and Arg-82 and Asp-85 on helix C. Helix G unfolds inside the bundle because the external force is applied to its C-terminal end in a direction perpendicular to the surface of the membrane. Inversely, helix F has to flip by 180 degrees to exit from the membrane because the applied force and the helical N-C axis point in opposite directions. At the highest peak of the force, which cannot be interpreted in single-molecule force spectroscopy experiments, helix F has a pronounced kink at Pro-186. Mutation of Pro-186 and/or the charged side chains mentioned above, which are involved in very favorable electrostatic interactions in the low-dielectric region of the membrane, are expected to reduce the highest peak of the force. Helices E and D unfold in a similar way to helices G and F, respectively. Hence, the force-distance profile and sequence of events during forced unfolding of bacterioopsin are influenced by the up-and-down topology of the seven-helix bundle. The sequential extraction of individual helices from the membrane suggests that the spontaneous (un) folding of bacterioopsin proceeds through metastable bundles of fewer than seven helices. The metastable states observed in the simulations provide atomic level evidence that corroborates the interpretation of very recent force spectroscopy experiments of bacteriorhodopsin refolding.
2006
- Single Amino Acid Contributions to Binding Affinity in Enzyme-Inhibitor Interactions: a Docking-Based Screening of BPTI-Beta Trypsin interaction
[Capitolo/Saggio]
D., Dell'Orco; DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
Enzyme-inhibitor interactions are crucial for normal functioning of many biological pathways. Point mutations in either the enzyme or the inhibitor molecule often lead to a modulation of the binding affinity (∆Go) with no major alterations in the 3D structure of the complex. The pos- sibility to screen in silico the effects of point mutations on ∆Go is of high interest, especially for protein design purposes. We have recently developed a computational protocol based on an existent rigid-body docking algorithm, which has shown a good capability to predict ∆Go changes upon mutations in protein-protein interactions. Here, we present the results obtained for the bovine pancreatic trypsin inhibitor (BPTI) and beta-Trypsin (β-Tryp) interaction. In this system, the BPTILys15 residue was replaced by eight different amino acids, hence varying the physico-chemical nature of the interface. The X-ray structure of each variant is available, as well as the effect of each substitution on the binding energetics. We compare our approach with data arising from both in vitro measurements and from another structure-based empirical approach, based on changes in solvent accessible surface areas (∆ASA) upon binding. The performance and the limitations of our approach are discussed.
2005
- Computational modeling approaches to Structure-Function Analysis of G Protein-Coupled Receptors
[Articolo su rivista]
Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Not available
2005
- Different structural requirements for the constitutive and the agonist-induced activities of the beta2-adrenergic receptor
[Articolo su rivista]
C., Ambrosio; P., Molinari; Fanelli, Francesca; Y., Chuman; M., Sbraccia; O., Ugur; T., Costa
abstract
We converted Ser-207, located in helix 5 of the beta2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to G(s) activation, we expressed the mutants in a G alpha(s)-tethered form. GTP exchange in such constructs is restricted to the fused alpha-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.
2005
- Probing fragment complementation by rigid-body docking: In silico reconstitution of calbindin D9k
[Articolo su rivista]
D., Dell'Orco; M., Seeber; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Fragment complementation is gaining an increasing impact as a nonperturbing method to probe noncovalent interactions within protein supersecondary structures. In this study, the fast Fourier transform rigid-body docking algorithm ZDOCK has been employed for in silico reconstitution of the calcium binding protein calbindin D9k, from its two EF-hands subdomains, namely, EF1 (residues 1-43) and EF2 (residues 44-75). The EF1 fragment has been used both in its wild type and in nine mutant forms, in line with in vitro experiments. Consistent with in vitro data, ZDOCK reconstituted the proper fold of wild-type and mutated calbindin, locating the nativelike structures (i.e., holding a root-mean-square deviation < 1 A with respect to the X-ray structure) among the first 10 top-scored solutions out of 4000. Moreover, the three independent in silico reconstitutions of wild-type calbindin ranked a nativelike structure at the top of the output list, that is, the best scored one. The algorithm has been also successfully challenged in reconstituting the EF2 homodimer from two identical copies of the monomer. Furthermore, quantitative models consisting of linear correlations between thermodynamic data and ZDOCK scores were built, providing a tested tool for very fast in silico predictions of the free energy of association of protein-protein complexes solved at the atomic level and known to not undergo significant conformational changes upon binding.
2005
- Rhodopsin activation follows precoupling with transducin: Inferences from computational analysis
[Articolo su rivista]
Fanelli, Francesca; D., Dell'Orco
abstract
The electrostatic and shape complementarities between the crystal structures of dark rhodopsin and heterotrimeric transducin (Gt) have been evaluated by exhaustively sampling the roto-translational space of one protein with respect to the other. Structural complementarity, reliability, and consistency with in vitro evidence all converge in the same rhodopsin-Gt complex, showing that the functionally important R135 of the E/DRY motif is almost accessible to the C-terminus of Gt(alpha) already in the dark state. The main inference from this study is that activation of rhodopsin and Gt may be concurrent processes, consisting of conformational changes in a supramolecular complex formed prior to the light-induced activation of the photoreceptor.
2005
- Structure-Function Relationships of the Luteinizing Hormone Receptor
[Articolo su rivista]
D., Puett; Y., Li; K., Angelova; G., Demars; T. P., Meehan; Fanelli, Francesca; P., Narayan
abstract
Of the 800-900 genes in the human genome that appear to encode G-protein-coupled receptors (GPCRs), two are known to encode receptors that bind the three heterodimeric human gonadotropins, luteinizing hormone (LH), chorionic gonadotropin (CG), and follicle-stimulating hormone (FSH). LH and CG bind to a common receptor, LHR, and FSH binds to a paralogous receptor. These GPCRs contain a relatively large ectodomain (ECD), responsible for high-affinity ligand binding, and a transmembrane portion, as in the other GPCRs. The ECD contains nine leucine-rich repeats capped by N-terminal and C-terminal cysteine-rich regions. The overall goal of this research is to elucidate the molecular mechanisms by which CG and LH bind to and activate LHR and the latter, in turn, activates Gs alpha. A combination of molecular modeling and site-directed mutagenesis, coupled with binding and signaling studies in transiently transfected HEK 293 cells expressing wild-type and mutant forms of LHR, has been used to develop and test models for the LHR ECD, the CG-LHR ECD complex, and the structural changes in the transmembrane helices and intracellular loops, particularly loop 2, that accompany receptor activation. In addition, a single-chain CG-LHR complex was designed in which a fusion protein of the two subunits of human CG was linked to full-length LHR. This ligand-receptor complex was shown to be constitutively active in cellular models and in transgenic mice, the latter of which exhibit precocious puberty. From a combination of molecular modeling, site-directed mutagenesis, genetic/protein engineering, and receptor characterization in cellular and animal models, considerable insight is being developed on the mechanisms of normal and aberrant activation of LHR.
2005
- The DRY motif as a molecular switch of the human oxytocin receptor
[Articolo su rivista]
N., Favre; Fanelli, Francesca; M., Missotten; J., Wilson; M., DI TIANI; C., Rommel; A., Scheer
abstract
The human oxytocin receptor is known to exhibit promiscuous activity by coupling to both Galpha(q) and Galpha(i) G proteins to activate distinct signaling pathways. A single-amino acid substitution within the highly conserved E/DRY motif at the cytosolic extension of helix 3 [i.e., D136(3.49)N] increased the rate of both basal and agonist-stimulated inositol phosphate (IP(3)) accumulation of the receptor. Furthermore, like for a typical constitutively active receptor, the partial agonist arginine vasopressin behaved as a full agonist for the D136(3.49)N mutant. Subsequently, both oxytocin and arginine vasopressin showed an increased potency in stimulating IP3 accumulation as compared to the wild-type receptor. Very interestingly, our experiments provide strong evidence that the D136(3.49)N mutant inhibits receptor signaling via Galpha(i)-mediated pathways while increasing the activity through the Galpha(q)-mediated pathways. Molecular simulations of the free and OT-bound forms of wild-type OTR and of the D136(3.49)N constitutively active mutant suggest that the receptor portions close to the E/DRY and NPxxY motifs are particularly susceptible to undergoing structural modification in response to activating mutations and agonist binding. Furthermore, computational modeling suggests that the OT-bound form of wild-type OTR is able to explore more states than the OT-bound form of the D136(3.49)N constitutively active mutant, consistent with its G protein promiscuity. Taken together, these observations emphasize the important role of the E/DRY motif not only in receptor activation but also in the promiscuity of G protein coupling. Knowledge of the mechanism of selective G protein coupling could aid drug discovery efforts to identify signaling specific therapies.
2005
- The formation of a salt bridge between helices 3 and 6 is responsible for the constitutive activity and lack of hormone responsiveness of the naturally occurring L457R mutation of the human lutropin receptor
[Articolo su rivista]
M., Zhang; D., Mizrachi; Fanelli, Francesca; D. L., Segaloff
abstract
The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.
2004
- Adenosine A(2A)-dopamine D-2 receptor-receptor heteromers. Targets for neuro-psychiatric disorders
[Articolo su rivista]
S., Ferre; F., Ciruela; M., Canals; D., Marcellino; J., Burgueno; V., Casado; J., Hillion; M., Torvinen; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe; S. R., Goldberg; M., Bouvier; K., Fuxe; Agnati, Luigi Francesco; C., Lluis; R., Franco; A., Woods
abstract
Emerging evidence show; that G protein-coupled receptors can form homo- and heteromers. These include adenosine A(2A) receptor-dopamine D-2 receptor heteromers, which are most probably localized in the dendritic spines of the striatopallidal GABAergic neurons, where they are in a position to modulate glutamatergic neurotransmission. The discovery of A(2A) receptor-dopamine D-2 receptor heteromers gives a frame for the well-known antagonistic interaction between both receptors, which is the bases for a new therapeutic approach for neuropsychiatric disorders, such as Parkinson´s disease and schizoprenia. The present review deals mainly with the biochemical and molecular aspects of A(2A) receptor-dopamine D-2 receptor interactions. Recent results at the molecular level show that A(2A) receptor-dopamine D-2 receptor heteromers represent the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization. Most probably A(2A) receptor-D-2 receptor heteromerization is not static, but subject to a dynamic regulation, related to the phosphorylation dependence of the A(2A) receptor epitope and to the ability of the D-2 receptor epitope to bind different partners. Finding out the mechanisms involved in this dynamic regulation can have important implications for the treatment of basal ganglia disorders, schizophrenia and drug addiction.
2004
- Erratum: Structural features of the inactive and active states of the melanin-concentrating hormone receiptors: Insights from molecular simulations (Proteins: Structure, Function and Genetics (2004) 56 (430-448))
[Articolo su rivista]
Vitale, R. M.; Pedone, C.; De Benedetti, P. G.; Fanelli, F.
abstract
2004
- Insight into mutation-induced activation of the luteinizing hormone receptor: Molecular simulations predict the functional behavior of engineered mutants at M398
[Articolo su rivista]
Fanelli, Francesca; M., VERHOEF POST; M., Timmerman; A., Zeilemaker; J. W. M., Martens; A. P. N., Themmen
abstract
In this study, molecular simulations have been combined with site-directed mutagenesis experiments to explore M398(2.43), a LH (lutropin) receptor (LHR) site in helix 2 susceptible to spontaneous activating mutations, and to develop a computational tool for predicting the functionality (i.e. active or nonactive) of LHR mutants.Site-directed mutagenesis experiments engineered 15 different substitutions for M389(2.43), which resulted in variable levels of constitutive activity, inversely correlated with the size of the replacing amino acid. This inverse correlation is suggested to be mediated by I460(3.46), M571(6.37), and Y623(7.53), the tyrosine of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the wild-type LHR between M398(2.43) and these three amino acids, resulting in structural modifications in the proximity to the E/DRY/W motif. An increment, above a threshold value, in the solvent accessibility of the cytosolic ends of helices 3 and 6 is the main structural feature shared by the active mutants of the LHR. This feature has been successfully used for predicting the functionality of the engineered mutants at M398(2.43), proving that molecular simulations can be useful for in silico screening of LHR mutants.
2004
- Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes
[Articolo su rivista]
S., Cotecchia; L., Stanasila; D., Diviani; K., Bjrklf; O., Rossier; Fanelli, Francesca
abstract
The aim of a large number of studies on G protein-coupled receptors was centered on understanding the structural basis of their main functional properties. Here, we will briefly review the results obtained on the alpha1-adrenergic receptor subtypes belonging to the rhodopsin-like family of receptors. These findings contribute, on the one hand, to further understand the molecular basis of adrenergic transmission and, on the other, to provide some generalities on the structure-functional relationship of G protein-coupled receptors.
2004
- Structural features of the inactive and active states of the melanin-concentrating hormone receptors: Insights from molecular simulations
[Articolo su rivista]
R. M., Vitale; C., Pedone; DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
Comparative molecular dynamics simulations of both subtypes 1 and 2 of the melanin-concentrating hormone receptor (MCHR1 and MCHR2, respectively) in their free and hormone-bound forms have been carried out. The hormone has been used in its full-length and truncated forms, as well as in 16 mutated forms. Moreover, MCHR1 has been simulated in complex with T-226296, a novel orally active and selective antagonist. The comparative analysis of an extended number of receptor configurations suggests that the differences between inactive (i.e., free and antagonist-bound) and active (i.e., agonist-bound) states of MCHRs involve the receptor portions close to the E/DRY and NPxxY motifs, with prominence to the cytosolic extensions of helices 2, 3, 6, and 7. In fact, the active forms of these receptors share the release of selected intramolecular interactions found in the inactive forms, such as that between R3.50 of the E/DRY motif and D2.40, and that between Y7.53 of the NPxxY motif and F7.60. Another feature of the active forms of both MCHRs is the approach of helix 8 to the cytosolic extension of helix 3. These features of the active forms are concurrent with the opening of a cleft at the cytosolic end of the helix bundle. For both MCHRs, the agonist-induced chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 6, following the ligand interaction with selected amino acids in the extracellular half of the receptor.
2004
- Synthesis, screening, and molecular modeling of new potent and selective antagonists at the alpha(1d) adrenergic receptor
[Articolo su rivista]
A., Leonardi; D., Barlocco; F., Montesano; G., Cignarella; G., Motta; R., Testa; E., Poggesi; M., Seeber; DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
In the present study, more than 75 compounds structurally related to BMY 7378 have been designed and synthesized. Structural variations of each part of the reference molecule have been introduced, obtaining highly selective ligands for the aid adrenergic receptor. The molecular determinants for selectivity at this receptor are essentially, held by the phenyl substituent in the phenylpiperazine moiety. The integration of an extensive SAR analysis with docking simulations using the rhodopsin-based models of the three alpha(1)-AR subtypes and of the 5-HT1A receptor provides significant insights into the characterization of the receptor binding sites as well as into the molecular determinants of ligand selectivity at the alpha(1d)-AR and the 5-HT1A receptors. The results of multiple copies simultaneous search (MCSS) on the substituted phenylpiperazines together with those of manual docking of compounds BAN 7378 and 69 into the putative binding sites of the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR, and the 5-HT1A receptors suggest that the phenylpiperazine moiety would dock into a site formed by amino acids in helices 3, 4, 5, 6 and extracellular loop 2, (E2), whereas the spirocyclic ring of the ligand docks into a site formed by amino acids of helices 1, 2, 3, and 7. This docking mode is consistent with the SAR data produced in this work. Furthermore, the binding site of the imide moiety does not allow for the simultaneous involvement of the two carbonyl oxygen atoms in H-bonding, interactions, consistent with the SAR data, in particular with the results obtained with the lactam derivative 128. The results of docking simulations also suggest that the second and third extracellular loops may act as selectivity filters for the substituted phenylpiperazines. The most potent and selective compounds for alpha(1d) adrenergic receptor, i.e., 69 (Rec 26D/038) and 128 (Rec 26D/073), are characterized by the presence of the 2,5-dichlorophenylpiperazine moiety.
2003
- Adenosine A(2A)-dopamine D2 receptor-receptor heteromerization - Qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer
[Articolo su rivista]
M., Canals; D., Marcellino; Fanelli, Francesca; F., Ciruela; DE BENEDETTI, Pier Giuseppe; Sr, Goldberg; K., Neve; K., Fuxe; Agnati, Luigi Francesco; As, Woods; S., Ferre; C., Lluis; M., Bouvier; R., Franco
abstract
There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively.
2003
- Constitutively active G protein-coupled receptor mutants: implications on receptor function and drug action
[Articolo su rivista]
S., Cotecchia; Fanelli, Francesca; T., Costa
abstract
Mutations of GPCRs can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis; others occur spontaneously in human diseases. The analysis of constitutively active GPCR mutants has attracted a large interest in the past decade, providing an important contribution to our understanding of the molecular mechanisms underlying receptor function and drug action.
2003
- Molecular dynamics simulations of the ligand-induced chemical information transfer in the 5-HT1A receptor
[Articolo su rivista]
M., Seeber; DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca
abstract
Comparative molecular dynamics simulations of the 5-HT1A receptor in its empty as well as agonist(i.e. active) and antagonist-bound (i.e. nonactive) forms have been carried out. The agonists 5-HT and (R)-8-OH-DPAT as well as the antagonist WAY100635 have been employed. The results of this study strengthen the hypothesis that the receptor portions close to the E/DRY/W motif, with prominence to the cytosolic extensions of helices 3 and 6, are particularly susceptible to undergo structural modification in response to agonist binding. Despite the differences in the structural/dynamics behavior of the two agonists when docked into the 5-HT1A receptor, they both exert a destabilization of the intrahelical and interhelical interactions found in the empty and antagonist-bound receptor forms between the arginine of the E/DRY sequence and both D133(3.49) and E340(6.30). For both agonists, the chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 61 following the ligand interaction with selected amino acids in the extracellular half of the receptor, such as D 116(3.32), S199(5.42), Y195(5.38), and F361(6.51). A significant reduction in the bend at P360(6.50), as compared to the empty and the antagonist-bound receptor forms, is one of the features of the agonist-bound forms that is related to the breakage of the interhelical salt bridge between the E/DRY arginine and E340(6.30). Another structural feature, shared by the agonist-bound receptor forms and not by the empty and antagonist-bound forms, is the detachment of helices 2 and 4, as marked by the movement of W161(4.50) away from helix 2, toward the membrane space.
2002
- A model for constitutive lutropin receptor activation based on molecular simulation and engineered mutations in transmembrane helices 6 and 7
[Articolo su rivista]
Angelova, K.; Fanelli, Francesca; Puett, D.
abstract
Many naturally occurring and engineered mutations lead to constitutive activation of the G protein-coupled lutropin receptor (LHR), some of which also result in reduced ligand responsiveness. To elucidate the nature of interhelical interactions in this heptahelical receptor and changes thereof accompanying activation, we have utilized site-directed mutagenesis on transmembrane helices 6 and 7 of rat LHR to prepare and characterize a number of single, double, and triple mutants. The potent constitutively activating mutants, D556(6.44)H and D556(6.44)Q, were combined with weaker activating mutants, N593(7.45)R and N597(7.49)Q, and the loss-of-responsiveness mutant, N593(7.45)A. The engineered mutants have also been simulated using a new receptor model based on the crystal structure of rhodopsin. The results suggest that constitutive LHR activation by mutations at Asp-556(6.44) is triggered by the breakage or weakening of the interaction found in the wild type receptor between Asp-556(6.44) and Asn-593(7.45). Whereas this perturbation is unique to the activating mutations at Asp-556(6.44), common features to all of the most active LHR mutants are the breakage of the charge-reinforced H-bonding interaction between Arg-442(3.50) and Asp-542(6.30) and the increase in solvent accessibility of the cytosolic extensions of helices 3 and 6, which probably participate in the receptor-G protein interface. Asn-593(7.45) and Asn-597(7.49) also seem to be necessary for the high constitutive activities of D556(6.44)H and D556(6.44)Q and for full ligand responsiveness. The new theoretical model provides a foundation for further experimental work on the molecular mechanism(s) of receptor activation.
2002
- Mutagenesis and modelling of the alpha(1b)-adrenergic receptor highlight the role of the helix 3/helix 6 interface in receptor activation
[Articolo su rivista]
Greasley, P.; Fanelli, Francesca; Rossier, O.; Abuin, L.; Cotecchia, S.
abstract
Computer simulations on a new model of the alpha1b-adrenergic receptor based on the crystal structure of rhodopsin have been combined with experimental mutagenesis to investigate the role of residues in the cytosolic half of helix 6 in receptor activation. Our results support the hypothesis that a salt bridge between the highly conserved arginine (R143(3.50)) of the E/DRY motif of helix 3 and a conserved glutamate (E289(6.30)) on helix 6 constrains the alpha1b-AR in the inactive state. In fact, mutations of E289(6.30) that weakened the R143(3.50)-E289(6.30) interaction constitutively activated the receptor. The functional effect of mutating other amino acids on helix 6 (F286(6.27), A292(6.33), L296(6.37), V299(6.40,) V300(6.41), and F303(6.44)) correlates with the extent of their interaction with helix 3 and in particular with R143(3.50) of the E/DRY sequence.
2002
- Structural Aspects of the Luteinizing Hormone Receptor: Information from Molecular Modeling and Mutagenesis
[Articolo su rivista]
Fanelli, Francesca; D., Puett
abstract
The luteinizing hormone receptor (LHR) is a member of the superfamily of G protein-coupled receptors and, in humans, binds two closely related ligands, members of the heterodimeric glycoprotein hormone family. This receptor is an essential component of the reproductive axis in males and females, and a number of naturally occurring pathophysiologic activating and inactivating mutations have been described. This review deals with the current state of knowledge of the structure of LHR based on molecular modeling and the supporting experimental data from engineered and naturally occurring mutations.
2002
- The alpha(1b)-adrenergic receptor subtype: Molecular properties and physiological implications
[Articolo su rivista]
S., Cotecchia; K., Bjrklf; O., Rossier; L., Stanasila; P., Greasley; Fanelli, Francesca
abstract
The aim of this review is to summarize some of the main findings from our laboratory as well as from others concerning the biochemical, molecular, and functional properties of the alpha1b-adrenergic receptor. Experimental and computational mutagenesis of the alpha1b-adrenergic receptor have been instrumental in elucidating some of the molecular mechanisms underlying receptor activation and receptor coupling to Gq. The knockout mouse model lacking the alpha1b-adrenergic receptor has highlighted the potential implication of this receptor subtype in variety of functions including the regulation of blood pressure, glucose homeostasis, and the rewarding response to drugs of abuse.
2002
- The lutropin/choriocrctnadotropin receptor, a 2002 perspective
[Articolo su rivista]
Ascoli, M.; Fanelli, Francesca; Segaloff, D.
abstract
Reproduction cannot take place without the proper functioning of the lutropin/choriogonadotropin receptor (LHR). When the LHR does not work properly, ovulation does not occur in females and Leydig cells do not develop normally in the male. Also, because the LHR is essential for sustaining the elevated levels of progesterone needed to maintain pregnancy during the first trimester, disruptions in the functions of the LHR during pregnancy have catastrophic consequences. As such, a full understanding of the biology of the LHR is essential to the survival of our species. In this review we summarize our current knowledge of the structure, functions, and regulation of this important receptor.
2002
- Understanding the mutation-induced activation of the lutropin receptor from computer simulation
[Relazione in Atti di Convegno]
Fanelli, Francesca
abstract
The luteinizing hormone receptor (LHR) is a member of the superfamily of G protein-coupled receptors and, in humans, binds two closely related ligands, members of the heterodimer glycoprotein hormone family. This receptor is an essential component of the reproductive axis in males and females and is particularly prone to spontaneous pathogenic activating and inactivating mutations.3D-model building and computer simulations have been employed to study the mutation-induced activation mechanism of the LHR.The results of molecular simulations on different LHR models converge into the hypothesis that the arginine of E/DRY/W sequence is an important switch of the LHR activation. They also suggest that a structural modification at the interface between the cytosolic extensions of helix 3 and helix 6 is important in the mutation-induced LHR activation and/or G protein recognition. The theoretical models provide insights into the structural features of the LHR sites susceptible to spontaneous activating mutations, constituting also useful tools for "in silico" prediction of the functional behaviour of LHR mutants.
2001
- Erratum: Pleiotropic effects of substitutions of a highly conserved leucine residue in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness (Molecular Endocrinology (2001) 15 (972-984))
[Articolo su rivista]
Shinozaki, H.; Fanelli, F.; Liu, X.; Jaquette, J.; Nakamura, K.; Segaloff, D. L.
abstract
2001
- Lutropin receptor function: Insights from natural, engineered, and computer-simulated mutations
[Articolo su rivista]
Fanelli, Francesca; A., Themmen; D., Puett
abstract
The lutropin receptor is a member of the superfamily of G protein-coupled receptors and, in humans, binds two closely related ligands, members of the heterodimer glycoprotein hormone family. As reviewed herein, comparative analyses of natural, engineered, and computer-simulated mutations of the lutropin receptor have provided considerable insight into the molecular basis of receptor function.
2001
- Mutational and computational analysis of the alpha-1b adrenergic receptor.Involvement of basic and hydrophobic residues in receptor activation and G protein coupling
[Articolo su rivista]
P. J., Greasley; Fanelli, Francesca; A., Scheer; L., Abuin; M., NENNINGER TOSATO; DE BENEDETTI, Pier Giuseppe; S., Cotecchia
abstract
To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.
2001
- Phenylpiperazinylalkylamino substituted pyridazinones as potent alpha(1) adrenoceptor antagonists
[Articolo su rivista]
D., Barlocco; G., Cignarella; V., Dal Piaz; M. P., Giovannoni; DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca; F., Montesano; E., Poggesi; A., Leonardi
abstract
QSAR models have been used for designing a series of compounds characterized by a N-phenylpiperazinylalkylamino moiety linked to substituted pyridazinones, which have been synthesized. Measurements of the binding affinities of the new compounds toward the alpha(1a)-, alpha(1b)-, and alpha(1d)-AR cloned subtypes as well as the 5-HT(1A) receptor have been done validating, at least in part, the estimations of the theoretical models. This study provides insight into the structure activity relationships of the alpha(1)-ARs ligands and their alpha(1)-AR/5-HT(1A) selectivity.
2001
- Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness
[Articolo su rivista]
Shinozaki, H.; Fanelli, Francesca; Liu, X.; Nakamura, K.; Segaloff, D. L.
abstract
It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.
2000
- Gonadotropin-independent precocious puberty due to luteinizing hormone receptor mutations in brazilian boys: a novel constitutively activating mutation in the first transmembrane helix
[Articolo su rivista]
A. C., Latronico; H., Shinozaki; G., GUERRA JR; M. A. A., Pereira; S. H. V. L., Marini; M. T. M., Baptista; I. J. P., Arnhold; Fanelli, Francesca; B. B., Mendonca; D. L., Segaloff
abstract
Naturally occurring activating mutations in the human LH receptor (hLHR) gene are the cause of sporadic or familial male gonadotropin-independent precocious puberty. We have previously reported three different activating mutations of the hLHR gene in four unrelated Brazilian boys with male-limited precocious puberty. In the current study, we examined three other Brazilian boys, two brothers and one unrelated boy, with gonadotropin-independent precocious puberty. Direct sequencing of the entire exon 11 of the hLHR gene in the two brothers revealed a heterozygous substitution of T for C at nucleotide 1103, resulting in the substitution of leucine at position 368 by proline in the first transmembrane helix. Their mother carried the same mutation, establishing the familial nature of this mutation. Human embryonic 293 cells expressing hLHR(L368P) bound hCG with the same high affinity as cells expressing the wild-type hLHR. Cells expressing the novel L368P mutation displayed up to a 12-fold increase in basal cAMP production compared with cells expressing the same number of cell surface wild-type hLHR, indicating constitutive activation of the mutant receptor. In addition, the cAMP levels in cells expressing the hLHR mutant were further augmented by hCG. Molecular dynamics simulations suggest that substitution of L368 of the hLHR by proline results in lack of a salt bridge interaction between D405 and R464 (distance 9. 0 A vs. 4.7 A in wild-type hLHR) as well as by the opening of a crevice between the second and third intracellular loops, which may allow G proteins greater accessibility. These structural features were shared by other activating mutants of the hLHR. Sequencing of exon 11 of the hLHR gene of the unrelated boy revealed that he carried a homozygous nucleotide substitution causing an A568V mutation in the third cytoplasmic loop of the receptor. This mutation was previously found in two unrelated Brazilian boys, but in heterozygous state. Clinical and hormonal data of the patient with the homozygous A568V were not different from those individuals with the Ala568Val mutation in a heterozygous state. Furthermore, the phenotype caused by dominant activating mutations of the hLHR gene are not altered when both alleles carry a mutant sequence. Our studies show that the A568V is the most frequent cause of male-limited precocious puberty in Brazilian boys. Lastly, the identification of a novel activating L368P mutation in the first transmembrane helix of two Brazilian boys with familial male-limited precocious puberty provides further insights into the mechanism of activation of the hLHR.
2000
- Molecular basis of ligand binding and receptor activation in the oxytocin and vasopressin receptor family
[Articolo su rivista]
B., Chini; Fanelli, Francesca
abstract
Although it is now widely accepted that G-protein-coupled receptors exist in at least two allosteric states, inactive and active, and that the spontaneous equilibrium between the two is regulated by various events including the binding of specific agonists and antagonists, the molecular counterparts of these functionally different states are still poorly understood. In this paper, we review our current knowledge concerning the structure-function relationships of the oxytocin and vasopressin receptors, focusing in particular on the process of receptor activation. Using a combined approach of site-directed mutagenesis and molecular modelling, we investigated the molecular events leading to agonist-dependent and -independent receptor activation in the human oxytocin receptor. Our analysis allows us to propose that the active conformations of this receptor are characterised by similar rearrangements of its cytosolic regions that ultimately lead to the opening of a putative docking site for the G-protein. Furthermore, the dynamics of these motions are similar to that observed in the alpha1B-adrenergic receptor, thus suggesting that, although activated by different ligands, the process of receptor isomerization in these two receptors is regulated by the same cluster of highly conserved residues and that common molecular events are responsible for receptor activation in different G-protein-coupled receptors.
2000
- Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation
[Articolo su rivista]
Scheer, A.; Costa, T.; Fanelli, Francesca; MHAOUTY KODJA, S.; Abuin, L.; NENNIGER TOSATO, M.; Cotecchia, S.; DE BENEDETTI, Pier Giuseppe
abstract
We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.
2000
- The ad hoc supermolecule approach to receptor ligand design
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca; Menziani, Maria Cristina; Cocchi, Marina
abstract
Among the ligand design methods based on the theoretical QSAR paradigm, the simple ad hoc supermolecule approach is presented and applied to a highly non-congeneric set of a1-adrenergic receptor antagonists. The performance of the approach is satisfactory and highlights its (semi)quantitative ligand design potentiality.
2000
- The alpha1a and alpha1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action
[Articolo su rivista]
S., Cotecchia; O., Rossier; Fanelli, Francesca; A., Leonardi; PIER G., DE BENEDETTI
abstract
In this chapter we summarize some aspects of the structure-functional relationship of the alpha 1a and alpha 1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the alpha 1a and alpha 1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies.
2000
- The α1a and α1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action
[Relazione in Atti di Convegno]
S., Cotecchia; O., Rossier; Fanelli, Francesca; A., Leonardi; DE BENEDETTI, Pier Giuseppe
abstract
In this chapter we summarize some aspects of the structure-functional relationship of the α 1a and α 1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the α 1a and α 1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies.
2000
- Theoretical investigation of substrate specificity for cytochromes p450 IA2, p450 IID6 and p450 IIIA4
[Articolo su rivista]
DE RIENZO, Francesca; Fanelli, Francesca; Menziani, Maria Cristina; DE BENEDETTI, Pier Giuseppe
abstract
Three-dimensional models of the cytochromes P450 IA2, P450 IID6 and P450 IIIA4 were built by means of comparative modeling using the X-ray crystallographic structures of P450 CAM, P450 BM-3, P450 TERP and P450 ERYF as templates. The three cytochromes were analyzed both in their intrinsic structural features and in their interaction properties with fifty specific and non-specific substrates. Substrate/enzyme complexes were obtained by means of both automated rigid and flexible body docking. The comparative analysis of the three cytochromes and the selected substrates, in their free and bound forms, allowed for the building of semi-quantitative models of substrate specificity based on both molecular and intermolecular interaction descriptors. The results of this study provide new insights into the molecular determinants of substrate specificity for the three different eukaryotic P450 isozymes and constitute a useful tool for predicting the specificity of new compounds.
2000
- Theoretical study on mutation-induced activation of the luteinizing hormone receptor
[Articolo su rivista]
Fanelli, Francesca
abstract
Here, three-dimensional model building and molecular dynamics simulations of the luteinizing hormone receptor have been employed to generate hypotheses about the molecular mechanisms underlying the activation of the receptor induced by naturally occurring activating mutations. The comparative analysis of the wild-type receptor and of 16 constitutively active or inactive mutants has been instrumental in inferring the structural/dynamic features which could characterize the inactive and the active forms of the receptor. These features have been also employed for predicting the functional behavior of new receptor mutants. The results of this study might provide a structural framework to interpret the pathological effects induced by mutations of the luteinizing hormone receptor. In addition, the proposed theoretical model could be useful for engineering new mutations or ligands able to modulate receptor function.
1999
- Activation mechanism of human oxytocin receptor: A combined study of experimental and computer-simulated mutagenesis
[Articolo su rivista]
Fanelli, Francesca; P., Barbier; D., Zanchetta; DE BENEDETTI, Pier Giuseppe; B., Chini
abstract
The aim of this study was to investigate the molecular changes associated with the transition of the human oxytocin receptor from its inactive to its active states. Mutation of the conserved arginine of the glutamate/aspartate-arginine-tyrosine motif located in the second intracellular domain gave rise to the first known constitutively active oxytocin receptor (R137A), whereas mutation of the aspartic acid located in the second transmembrane domain led to an inactive receptor (D85A). The structural features of the constitutively active and inactive receptor mutants were compared with those of the wild type in its free and agonist-bound states. The results suggest that, although differently triggered, the activation process induced by the agonist and the activating mutation are characterized by the opening of a solvent exposed site formed by the 2nd intracellular loop, the cytosolic extension of helix 5, and the 3rd intracellular loop; on the contrary, the D85A mutation prevents oxytocin from triggering the opening of a cytosolic site. On the basis of these findings, we hypothesize that this cytosolic crevice plays an important role in G protein recognition. Finally, comparative analysis of the free- and agonist-bound forms of the wild-type oxytocin receptor and alpha(1B) adrenergic receptor suggests that the highly conserved polar amino acids and the seven helices play similar mechanistic roles in the different G protein-coupled receptors.
1999
- Constitutively active receptor mutants as probes for studying the mechanisms underlying G protein-coupled receptor activation
[Capitolo/Saggio]
S., Cotecchia; Fanelli, Francesca; A., Scheer; DE BENEDETTI, Pier Giuseppe
abstract
In this chapter, we illustrate a strategy to explore the potential molecular changes correlated with the transition from R to R* using the Gq-coupled alpha1B-AR as model system.
1999
- Structure-function relationships of the alpha(1b)-adrenergic receptor
[Articolo su rivista]
A., Scheer; Fanelli, Francesca; D., Diviani; DE BENEDETTI, Pier Giuseppe; S., Cotecchia
abstract
The alpha1b-adrenergic receptor (AR) is a member of the large superfamily of seven transmembrane domain (TMD) G protein-coupled receptors (GPCR). Combining site-directed mutagenesis of the alpha1b-AR with computational simulations of receptor dynamics, we have explored the conformational changes underlying the process of receptor activation, i.e. the transition between the inactive and active states. Our findings suggest that the structural constraint stabilizing the alpha1b-AR in the inactive form is a network of H-bonding interactions amongst conserved residues forming a polar pocket and R143 of the DRY sequence at the end of TMDIII. We have recently reported that point mutations of D142, of the DRY sequence and of A293 in the distal portion of the third intracellular loop resulted in ligand-independent (constitutive) activation of the alpha1b-AR. These constitutively activating mutations could induce perturbations resulting in the shift of R143 out of the polar pocket. The main role of R143 may be to mediate receptor activation by triggering the exposure of several basic amino acids of the intracellular loops towards the G protein. Our investigation has been extended also to the biochemical events involved in the desensitization process of alpha1b-AR. Our results indicate that immediately following agonist-induced activation, the alpha1b-AR can undergo rapid agonist-induced phosphorylation and desensitization. Different members of the G protein coupled receptor kinase family can play a role in agonist-induced regulation of the alpha1b-AR. In addition, constitutively active alpha1b-AR mutants display different phosphorylation and internalization features. The future goal is to further elucidate the molecular mechanism underlying the complex equilibrium between activation and inactivation of the alpha1b-AR and its regulation by pharmacological substances. These findings can help to elucidate the mechanism of action of various agents displaying properties of agonists or inverse agonists at the adrenergic system.
1999
- Theoretical study on receptor-G protein recognition: new insights into the mechanism of the α1b-adrenergic receptor activation
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; A., Scheer; S., Cotecchia; DE BENEDETTI, Pier Giuseppe
abstract
Theoretical study on receptor/G protein recognition: new insights into the mechanism of the α1b-adrenergic receptor activation.
1999
- Theoretical study on the electrostatically driven step of receptor-G protein recognition
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; A., Scheer; Cotecchia, S.; DE BENEDETTI, Pier Giuseppe
abstract
Theoretical study on the electrostatically driven step of receptor-G protein recognition
1999
- nverse agonism and neutral antagonism at alpha(1a)- and alpha(1b)-adrenergic receptor subtypes
[Articolo su rivista]
O., Rossier; L., Abuin; Fanelli, Francesca; A., Leonardi; S., Cotecchia
abstract
We have characterized the pharmacological antagonism, i.e., neutral antagonism or inverse agonism, displayed by a number of alpha-blockers at two alpha1-adrenergic receptor (AR) subtypes, alpha(1a)- and alpha(1b)-AR. Constitutively activating mutations were introduced into the alpha(1a)-AR at the position homologous to A293 of the alpha(1b)-AR where activating mutations were previously described. Twenty-four alpha-blockers differing in their chemical structures were initially tested for their effect on the agonist-independent inositol phosphate response mediated by the constitutively active A271E and A293E mutants expressed in COS-7 cells. A selected number of drugs also were tested for their effect on the small, but measurable spontaneous activity of the wild-type alpha(1a)- and alpha(1b)-AR expressed in COS-7 cells. The results of our study demonstrate that a large number of structurally different alpha-blockers display profound negative efficacy at both the alpha(1a)- and alpha(1b)-AR subtypes. For other drugs, the negative efficacy varied at the different constitutively active mutants. The most striking difference concerns a group of N-arylpiperazines, including 8-[2-[4-(5-chloro-2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4, 5] decane-7,9-dione (REC 15/3039), REC 15/2739, and REC 15/3011, which are inverse agonists with profound negative efficacy at the wild-type alpha(1b)-AR, but not at the alpha(1a)-AR.
1998
- Ab initio modeling and molecular dynamics simulation of the alpha 1b-adrenergic receptor activation
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; A., Scheer; S., Cotecchia; DE BENEDETTI, Pier Giuseppe
abstract
This work describes the ab initio procedure employed to build an activation model for the alpha(1b)-adrenergic receptor (alpha(1b)-AR). The first version of the model was progressively modified and complicated by means of a many-step iterative procedure characterized by the employment of experimental validations of the model in each upgrading step. a combined simulated (molecular dynamics) and experimental mutagenesis approach was used to determine the structural and dynamic features characterizing the inactive and active states of alpha(1b)-AR. The latest version of the model has been successfully challenged with respect to its ability to interpret and predict the functional properties of a large number of mutants. The iterative approach employed to describe alpha(1b)-AR activation in terms of molecular structure and dynamics allows further complications of the model to allow prediction and interpretation of an ever-increasing number of experimental data.
1998
- Antagonist profile and molecular dynamic simulation of a Drosophila melanogaster muscarinic acetylcholine receptor
[Articolo su rivista]
C. M., Reaper; Fanelli, Francesca; S. D., Buckingham; N. S., Millar; D. B., Sattelle
abstract
A stably-transfected, Drosophila cell line (S2-DMl-1) expressing the Drosophila DMl muscarinic acetylcholine receptor (mAChR) exhibits high-affinity, saturable, specific binding of the radiolabelled muscarinic antagonist [3H]-N-methyl scopolamine ([3H]-NMS) with an equilibrium dissociation constant (Kd) of 0.67 +/- 0.02 and a Bmax of 1.53 +/- 0.3 pmol/mg protein. Displacement of [3H]-NMS by mAChR antagonists results in the pharmacological profile: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > hexahydrosiladifenidol > p-fluorohexahydrosiladifenidol > nitrocaramiphen > pirenzepine > methoctramine > AFDX-116. This antagonist profile most closely resembles that of the vertebrate M3 mAChR subtype. In this study, however, we have demonstrated that the antagonist profile of DM1 is distinct from those of vertebrate mAChR subtypes. Molecular dynamic simulations of the Drosophila muscarinic receptor are presented in the free, carbamylcholine-bound and NMS-bound forms. Theoretical, quantitative structure-activity relationship models have been developed; a good correlation is observed between the interaction energies of the minimized ligand-receptor complexes and the pharmacological affinities of the antagonists tested.
1998
- Computer modeling of size and shape descriptors of alpha 1-adrenergic receptor antagonists and quantitative structure-affinity/selectivity relationships
[Articolo su rivista]
Montorsi, Monia; Menziani, Maria Cristina; Cocchi, Marina; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Computational chemistry and molecular modeling procedures allow us to define and compute ad hoc size and shape descriptors on the different prototropic forms assumed by drugs in biotest solutions. Together with experimental data measured on a well-identified target receptor, these descriptors are essential elements for obtaining simple, consistent, comparable, and easily interpretable theoretical quantitative structure-activity relationship (QSAR) models based on the ligand similarity-target receptor complementarity paradigm. In this context, quantitative size and shape affinity/subtype selectivity relationships have been modeled for a large set of very heterogeneous alpha(1a)-, alpha(1b)-, and alpha(1d)-adrenergic receptor antagonists. The linear QSAR models generated have been validated by predicting both binding affinity and selectivity of a test set of noncongeneric antagonists. The satisfactory results obtained highlight both the simplicity and the versatility of the approach presented.
1998
- Hydrophobicity of residue(351) of the G protein G(i1)alpha determines the extent of activation by the alpha(2A)-adrenoceptor
[Articolo su rivista]
D. S., Bahia; A., Wise; Fanelli, Francesca; M., Lee; S., Rees; G., Milligan
abstract
Cysteine351 is the site for pertussis toxin-catalyzed ADP-ribosylation in the G protein Gi1 alpha. Alteration of this residue, or the equivalent cysteine in other Gi-family G proteins, has been used to examine specific interactions between receptors and these G proteins. However, no systematic analysis has been performed to determine the quantitative effect of such alterations. To address this we mutated cysteine351 of Gi1 alpha to all other possible amino acids. Each of the G protein mutants was transiently coexpressed along with the porcine alpha 2A-adrenoceptor in HEK 293/T cells. Following pertussis toxin treatment of the cells, membranes were prepared and the capacity of the agonist UK14304 to stimulate the binding of [35S]GTP gamma S to the modified G proteins was measured. A spectrum of function was observed. The presence of either a charged amino acid or a proline at this position essentially attenuated agonist regulation. The wild-type G protein did not result in maximal stimulation by agonist. The presence of certain branched chain aliphatic amino acids or bulky aromatic R groups at amino acid351 resulted in substantially greater maximal stimulation by the alpha 2A-adrenoceptor than that achieved with the wild-type sequence. The degree of activation of the forms of Gi1 alpha correlated strongly with the octanol/water partition coefficient of the amino acid at residue351. Variation in EC50 values for agonist-induced stimulation of binding of [35S]GTP gamma S to the mutant G proteins also correlated with the octanol/water partition coefficient. These results define a central role for hydrophobicity of this residue in defining productive receptor-G protein interactions.
1998
- IIsoxazolo-[3,4-d]-pyridazin-7-(6H)-ones and their corresponding 4,5-disubstituted-3-(2H)-pyridazinone analogues as new substrates for alpha1-adrenoceptor selective antagonists: synthesis, modeling, and binding studies
[Articolo su rivista]
F., Montesano; D., Barlocco; V., DAL PIAZ; A., Leonardi; E., Poggesi; Fanelli, Francesca; P. G., DE BENEDETTI
abstract
A series of phenylpiperazinylalkyl moieties were attached to monocyclic or bicyclic substituted pyridazinones and the new compounds tested for their affinity towards alpha1-adrenoceptor and its alpha1a, alpha1b and alpha1d subtypes, as well as serotonin 5-HT1A receptor. Several analogues (5, 6, 9, and 10) showed remarkable potency and selectivity towards alpha1a, and alpha1d with respect to alpha1b subtype. None of the test compounds exhibited significant affinity for 5-HT1A receptor. Finally, on the basis of the alpha1-AR subtypes 3D models recently proposed, we have elaborated theoretical interaction models for the new compounds. The theoretical study allowed us to predict the affinity of the new compounds as well as to infer the structural/dynamics determinants of their interaction with the three alpha1-AR subtypes.
1998
- Identification of a constitutively active mutant of the human oxytocin receptor
[Relazione in Atti di Convegno]
B., Chini; E., Albertazzi; P. G., DE BENEDETTI; Fanelli, Francesca
abstract
Abstract not present
1998
- Molecular mechanisms involved in the activation and regulation of the alpha 1-adrenergic receptor subtypes
[Articolo su rivista]
S., Cotecchia; A., Scheer; D., Diviani; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
The adrenergic receptors (ARs) belong to the superfamily of membrane-bound G protein coupled receptors (GPCRs). Our investigation has focused on the structure-function relationship of the alpha 1b-AR subtype used as the model system for other GPCRs. Site-directed mutagenesis studies have elucidated the structural domains of the alpha 1b-AR involved in ligand binding, G protein coupling or desensitization. In addition, a combined approach using site-directed mutagenesis and molecular dynamics analysis of the alpha 1b-AR has provided information about the potential mechanisms underlying the activation process of the receptor, i.e. its transition from the 'inactive' to the 'active' conformation.
1997
- Alpha 1-adrenoceptor subtype selectivity: Molecular modelling and theoretical quantitative structure-affinity relationships
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Fanelli, Francesca; Menziani, Maria Cristina; Cocchi, Marina; R., Testa; A., Leonardi
abstract
This study constitutes a preliminary rationalization, at the molecular level, of antagonist selectivity towards the three cloned alpha 1-adrenergic receptor (alpha 1-AR) subtypes. Molecular dynamics simulations allowed a structural/dynamics analysis of the seven alpha-helix-bundle models of the bovine alpha 1a-, hamster alpha 1b-, and rat alpha 1d-AR subtypes. The results showed that the transmembrane domains of these subtypes have different dynamic behaviours and different topographies of the binding sites, which are mainly constituted by conserved residues. In particular, the alpha 1a-AR binding site is more flexible and topographically different with respect to the other two subtypes. The results of the theoretical structural/dynamics analysis of the isolated receptors are consistent with the binding affinities of the 16 antagonists tested towards the three cloned alpha 1-AR subtypes. Moreover, the theoretical quantitative structure-affinity relationships obtained from the antagonist-receptor interaction models further corroborates the hypothesis that selectivity towards one preferential subtype is mainly modulated by receptor and/or ligand distortion energies. In other words, subtype selectivity seems to be mainly guided by the dynamic complementarity (induced fit) between ligand and receptor. On the basis of the quantitative models presented it is possible to predict both affinities and selectivities of putative alpha 1-AR ligands as well as to estimate the theoretical alpha 1-AR subtype affinities and selectivities of existing antagonists.
1997
- Conformational analysis and theoretical quantitative size and shape-affinity relationships of N-4-protonated N-1-arylpiperazine 5-HT1A serotoninergic ligands
[Articolo su rivista]
Cocchi, Marina; Fanelli, Francesca; Menziani, Maria Cristina; DE BENEDETTI, Pier Giuseppe
abstract
Conformational analysis for 24 arylpiperazines in their neutral and N-4-protonated forms has been performed in the AM1 framework. Both these derivatives and eight reference compounds considered in this study are ligands of the 5-HT1A serotoninergic receptor. Quantum chemical reactivity indices, solvation free energies (AMSOL 5.0) and molecular modelling derived ad hoc size and shape descriptors have been computed and correlated with the literature 5-HT1A binding affinity data values. The quantitative size-shape affinity relationships obtained confirm the validity and versatility of the ad hoc descriptors employed. A different role has been postulated for the neutral and protonated forms of the arylpiperazines considered in the molecular recognition process of the 5-HT1A receptor binding site.
1997
- Constitutively active α1B-adrenergic receptor mutants: potential mechanisms underlying receptor activation
[Relazione in Atti di Convegno]
A., Scheer; Fanelli, Francesca; T., Costa; DE BENEDETTI, Pier Giuseppe; S., Cotecchia
abstract
Not Available
1997
- The activation process of the alpha(1B)-adrenergic receptor: Potential role of protonation and hydrophobicity of a highly conserved aspartate
[Articolo su rivista]
Scheer, A; Fanelli, Francesca; Costa, T.; DE BENEDETTI, P. G.; Cotecchia, S.
abstract
In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the alpha1B-adrenergic receptor (alpha1B-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the alpha1B-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.
1997
- Theoretical investigation of IL-6 multiprotein receptor assembly
[Articolo su rivista]
Menziani, Maria Cristina; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Interleukin-6 (IL-6) is a multifunctional cytokine that regulates cell growth, differentiation, and cellular functions in many cell lineages, Recently, evidences for the formation of an active hexameric complex with an IL-6:IL-6R alpha:gp130 stoichiometry of 2:2:2 have been obtained by different experimental approaches, Analysis of the electrostatic potential complementarity between IL-6 and its receptors has been used, in this study to guide the assembly of homology-based 3D models of the components, The results strongly support a mechanism whereby the active cytokine (IL-6: IL-6R alpha) associates with the signal transducing gp130 protein, and the trimeric complex formed further dimerizes to form the hexameric species. Furthermore, computational simulations of the multiprotein complexes provide a rationalization of data from mutation experiments and highlight some key protein-protein interactions which have not yet been the subject of mutagenesis studies.
1997
- Thienocycloheptapyridazines as new muscarinic agents
[Articolo su rivista]
D., Barlocco; G., Cignarella; Fanelli, Francesca; B., Vitalis; P., Matyus; DE BENEDETTI, P. G.
abstract
A series of thienocycloheptapyridazines (3aa-dd), structurally related to Minaprine, was synthesized and compounds tested for their affinity towards muscarinic receptors. All of them showed Ki values in the micromolar range towards both the antagonist 3H-QNB and the agonist 3H-OXO-M, thus indicating that they act as antagonists at the muscarinic receptors. Moreover a theoretical study was performed on their interaction behaviour with a three dimensional (3-D) model of the human m1 muscarinic receptor.
1996
- 2-(Substituted)amino-2,8-diazaspiro[4,5]decan-1,3-diones as potential muscarinic agonists: synthesis, modeling and binding studies
[Articolo su rivista]
D., Barlocco; Fanelli, Francesca; G., Cignarella; S., Villa; F., Cattabeni; W., Balduini; M., Cimino; P. G., DE BENEDETTI
abstract
A series of 2-(acyl)amino-8-substituted-2,8-diazaspiro[4,5]decan-1,3-diones (5a-j), structurally related to the muscarinic agonist RS-86, was synthesized and compounds tested for their affinity towards muscarinic receptors. Though all compounds proved to be less active than the model in binding studies, only three derivatives (5a, b, c) being able to significantly displace 3H-QNB at mM concentration, their behaviour could be interpreted in terms of theoretical molecular descriptors computed on the basis of the suggestions coming from Molecular Dynamics simulations of ligand-receptor complexes.
1996
- Amino acids of the α1B-adrenergic receptor involved in agonist binding: further differences in docking catecholamines to receptor subtypes
[Articolo su rivista]
A., Cavalli; Fanelli, Francesca; C., Taddei; P. G., DE BENEDETTI; S., Cotecchia
abstract
Site-directed mutagenesis and molecular dynamics analysis of the 3-D model of the alpha1B-adrenergic receptor (AR) were combined to identify the molecular determinants of the receptor involved in catecholamine binding. Our results indicate that the three conserved serines in the fifth transmembrane domain (TMD) of the alpha1B-AR play a distinct role in catecholamine binding versus receptor activation. In addition to the amino acids D125 in TMDIII and S207 in TMDV directly involved in ligand binding, our findings identify a large number of polar residues playing an important role in the activation process of the alpha1B-AR thus providing new insights into the structure/function relationship of G protein-coupled receptors.
1996
- Computational simulations of stem-cell factor c-Kit receptor interaction
[Articolo su rivista]
Menziani, Maria Cristina; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Stem-cell factor (SCF) is a noncovalent homodimeric cytokine that exhibits profound biological function in the early stages of hematopoiesis by binding to a cell surface tyrosine kinase receptor that is encoded by the c-Kit proto-oncogene. The results obtained from a combined implementation of homology-based molecular modeling and computational simulations in the study of species-specific SCF/c-Kit interactions are reported. The structural models of the human and rat SCF Ligands are based on the close structural similarity to the cytokine M-CSF, whose C alpha structure has recently become available. The constant domains of the human Fc fragment are used as a template for the ligand binding domains of the c-Kit receptor. The factors responsible for the stabilization of the SCF quaternary structure and the molecular determinants for ligand recognition and ligand specificity have been identified by assessing the conformational, topographical, and dynamic features of the isolated ligands and of the ligand-receptor complexes.
1996
- Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation
[Articolo su rivista]
Scheer, A.; Fanelli, Francesca; Costa, T.; DE BENEDETTI, P. G.; Cotecchia, S.
abstract
Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.
1996
- Molecular mechanisms underlying the activation and regulation of the alpha 1B-adrenergic receptor
[Articolo su rivista]
S., Cotecchia; A., Scheer; Fanelli, Francesca; T., Costa
abstract
Abstract not present
1996
- The Heuristic-Direct approach to Quantitative Structure-Activity Relationship Analysis of Ligand-G Protein Coupled Receptor Complexes
[Capitolo/Saggio]
Menziani, Maria Cristina; Fanelli, Francesca; Cocchi, Marina; DE BENEDETTI, Pier Giuseppe
abstract
The Heuristic-Direct approach to Quantitative Structure-Activity Relationship Analysis of Ligand-G Protein Coupled Receptor Complexes
1995
- Comparative molecular dynamics study of the seven-helix bundle arrangement of G-protein coupled receptors
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; Cocchi, Marina; DE BENEDETTI, Pier Giuseppe
abstract
A comparative molecular dynamics study has been performed on the seven-helix bundle arrangement of seven G-protein coupled receptors (GPCRs). They are hamster alpha(1B)-, human alpha(2)-, beta(2)-adrenergic, human D-2-dopaminergic, human 5-HT1A-serotoninergic, human ml-muscarinic receptors and bovine rhodopsin. Starting from a rhodopsin-like input structure and a bacteriorhodopsin-like input structure, a similar arrangement of the averaged helix bundles was obtained. This may be due to the topography of some fundamental polar positions in both the input structures which is substantially the same and dictates, through the establishment of similar H-bonding networks, the helix-helix packing. By comparing the averaged structures and the packing interaction parameters obtained for the GPCRs considered with that of bacteriorhodopsin, we observe that the GPCRs share a similar packing arrangement of their transmembrane helix bundles which differs from that of bacteriorhodopsin. The results obtained from the quantitative and comparative molecular modelling of the GPCRs constitute an important preliminary step in a general understanding of both structure-function and structure activity-selectivity relationships in these proteins, at the molecular level.
1995
- Computer simulations of signal transduction mechanism in alpha 1B-adrenergic and m3-muscarinic receptors
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; DE BENEDETTI, Pier Giuseppe
abstract
Molecular dynamics simulations of the hamster alpha(1B)-adrenergic and the rat m3-muscarinic seven-helix bundle receptor models have been carried out. The free, agonist-bound and antagonist-bound forms have been considered, Moreover, three mutant forms of the m3-muscarinic receptor (N507-->A, N507-->D and N507-->S) have also been simulated; among these, the N507-->S mutant shows a constitutive activity, A comparative structural/dynamics analysis has been performed to elucidate (i) the perturbations induced by the functionally different ligands upon binding to their target receptor, (ii) the features of the three single-point mutants with respect to the receptor wild type and (iii) the properties shared by the agonist-bound forms of the alpha(1B)-adrenergic receptor and the m3-muscarinic receptor and by the constitutively active mutant N507-->S. The consistency obtained between the structural rearrangement of the transmembrane seven-helix bundle models considered, and the experimental pharmacological efficacies of the ligands and of the mutants, constitute an important validation of the 3-D models obtained and allow the inference of the mechanism of ligand- or mutation-induced receptor activation at the molecular level.
1995
- Molecular dynamics simulations of m3-muscarinic receptor activation and QSAR analysis
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; DE BENEDETTI, Pier Giuseppe
abstract
Molecular dynamics simulations of the rat m3-muscarinic seven-helix-bundle receptor models were performed on the free, agonist-bound and antagonist-bound forms. A comparative structural/dynamics analysis was performed in order to explain the perturbations induced by the functionally different ligands when binding to their target receptor. Theoretical quantitative structure-activity relationship models were developed; a good correlation was obtained between the interaction energies of the minimized average ligand-receptor complexes and the pharmacological affinities of the considered ligands. The consistency obtained between the structural rearrangement of the transmembrane seven-helix-bundle models considered and the experimental pharmacological efficacies and affinities of the ligands constitutes an important validation of the 3-D models proposed and allows the inference of the mechanism of ligand-induced or mutation-induced receptor activation at the molecular level.
1995
- Prototropic molecular forms and theoretical descriptors in QSAR analysis
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Cocchi, Marina; Fanelli, Francesca
abstract
Computational chemistry allows us to define and compute ad hoc theoretical reactivity and size-shape descriptors on the different prototropic forms assumed by drugs in pharmacological test solutions. These are essential elements for obtaining simple, consistent, comparable and easily interpretable theoretical QSAR models based on the ligand similarity-target complementarity paradigm. In this context, theoretical QSAR models have been obtained for 34 structurally and pharmacologically (antagonists, partial agonists and full agonists) heterogeneous M(1)-muscarinic ligands and the above concepts have been highlighted.
1995
- Quantitative structure-affinity/selectivity relationship analysis on three-dimensional models of the complexes between the ETA and ETB receptors and C-terminal endothelin hexapeptide antagonists
[Articolo su rivista]
Menziani, Maria Cristina; Cocchi, Marina; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
The application of molecular modelling and regression analysis techniques to the complexes between the ET(A) and ET(B) receptors and a series of C-terminal endothelin hexapeptide antagonists allowed the identification of the structural determinants for recognition and the quantitative elucidation of the receptor structure-affinity/selectivity relationships.
1995
- The Heuristic-Direct Approach to QSAR Analysis of Ligand-G-Protein Coupled Receptor Complexes
[Capitolo/Saggio]
DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Fanelli, Francesca; Cocchi, Marina
abstract
Not available
1995
- Theoretical QSAR analysis on three dimensional models of the complexes between peptide and non-peptide antagonists with the ETA and ETB receptors
[Capitolo/Saggio]
Menziani, Maria Cristina; Cocchi, Marina; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Not available
1995
- Theoretical quantitative structure-activity relationship analysis of congeneric and non-congeneric α1-adrenoceptor antagonists: a chemometric study
[Articolo su rivista]
Cocchi, Marina; Menziani, Maria Cristina; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Molecular orbital calculations (AM1) and molecular modelling procedures (QUANTA/CHARMm) have been performed both on a congeneric (prazosin analogs) and on a non-congeneric series of alpha(1)-adrenergic antagonists. A large variety of theoretical molecular descriptors has been obtained and compared by principal component analysis (PCA). The generating optimal least squares estimations (GOLPE) procedure has been used to derive quantitative structure-activity relationships (QSARs). Good predictive QSAR models with a restricted pool of informative theoretical descriptors have been obtained. These results support the generality of the theoretical QSAR approach proposed; in fact both congeneric and non-congeneric molecular series were satisfactorily modeled. Moreover, the high and well-defined physical information content encoded in the theoretical descriptors considered allows the rationalization of the structural heterogeneity of the molecules examined as differences in the complementary intermolecular interactions of the studied ligands towards their common receptor.
1994
- The heuristic-direct approach to theoretical quantitative structure activity relationship analysis of α1- adrenoceptor ligands
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; Cocchi, Marina; A., Leonardi; DE BENEDETTI, Pier Giuseppe
abstract
The heuristic-direct quantitative structure-activity relationship approach has been applied to fifteen non-congeneric alpha1-adrenergic receptor (alpha1-AR) ligands interacting with the rat alpha1A/D-AR subtype. The good linear correlations, which have been obtained between calculated binding energies and the pharmacological affinities, allow one to predict the pharmacological affinity of new ligands. Moreover, according to the alpha1A/D-receptor model proposed, it has been possible to speculate on the amino acid residues which are mainly involved in the interaction with the ligands. This novel procedure consitutes a powerful tool for the design of new selective leads based on explicit intermolecular interactions and for suggesting site-directed mutagenesis studies, in order to give, iteractively, further support and improvement to the predictive and interpretative aspects of the model.
1994
- Theoretical Quantitative Structure-Activity Relationship Analysis on Three Dimensional Models of Ligand-m1 Muscarinic Receptor Complexes
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; A., Carotti; P. G., DE BENEDETTI
abstract
The heuristic-direct QSAR (quantitative structure-activity relationships) approach has been applied to a series of 34 muscarinic receptor ligands, including antagonists, weak partial agonists, partial agonists and full agonists, interacting with the human ml-muscarinic receptor subtype. The first step of this procedure consists of the computer-aided 3D-model building of the receptor. The second step involves docking simulations with selected ligands, maximizing the complementarity between ligand and receptor. In the third step, a detailed and extensive correlation analysis between the computed interaction energies, their components and the experimental pharmacological affinity and action is accomplished in order to evaluate the consistency of the QSAR model proposed and to provide a quantitative tool for comparisons among the different complexes considered. In this context, good linear correlations have been obtained between ad hoc theoretical intermolecular interaction descriptors and the pharmacological action, which allow one to classify quantitatively and predict the pharmacological action of new ligands. Finally, according to the ml-receptor model proposed, it has been possible to speculate on the amino acid residues which are mainly involved in the interaction with the ligands, and on the nature of the prevailing intermolecular interactions which are responsible for the different behaviour of antagonists, weak partial agonists, partial agonists and full agonists.
1994
- Theoretical quantitative size and shape activity and selectivity analyses of 5-HT1A serotonin and α1-adrenergic receptor ligands
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Cocchi, Marina; Menziani, Maria Cristina; Fanelli, Francesca
abstract
Quantum chemical reactivity indices and molecular modelling derived size and shape descriptors have been computed for 18 5-HT1A serotonin and alpha1-adrenergic receptor ligands. The quantitative size shape affinity selectivity relationships obtained support the general validity and versatility of the ad hoc size shape descriptors employed.
1993
- Enantiomeric resolution of sulfoxides on a DACH‐DNB chiral stationary phase: A quantitative structure–enantioselective retention relationship (QSERR) study
[Articolo su rivista]
C., Altomare; A., Carotti; S., Cellamare; Fanelli, Francesca; F., Gasparrini; C., Villani; P. A., Carrupt; B., Testa
abstract
The interaction mechanism of a variety of racemic alkyl aryl sulfoxides with a pi-acid HPLC stationary phase containing N,N'-(3,5-dinitrobenzoyl)-trans-1, 2-diaminocyclohexane chiral selector was investigated by means of quantum-chemical calculations (MNDO), partial least squares (PLS) analysis, and 3D comparative molecular field analysis (CoMFA). Quantitative structure-enantioselective retention relationships (QSERR), were derived which have yielded significant insights into physicochemical properties primarily responsible for chiral recognition. The increase in retention (k') is favored especially by the analyte pi-basic character, accounted for by the sum of the electrophilic superdelocalizabilities of all aromatic carbon atoms (S(Ph)HOMO), and to minor extent by the H-bond basicity of the sulfoxide oxygen and the hydrophilicity of solutes. In contrast, the separation factor (alpha) varied mainly with the steric properties of the substituents and with polar and electrostatic properties of the sulfoxide group. A 3D-QSERR analysis using CoMFA methodology has provided a more complete description of factors responsible for chiral recognition and has proven to be a useful tool to examine differences in noncovalent fields (both the electrostatic and the steric) mostly associated with variations of enantioselectivity.
1993
- The heuristic-direct approach to quantitative structure-activity relationship analysis
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Fanelli, Francesca; Cocchi, Marina
abstract
The heuristic-direct approach to quantitative structure-activity relationship analysis
1993
- Theoretical quantitative structure-activity analysis and pharmacophore modelling of selective non congeneric α1a-adrenergic antagonists
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Cocchi, Marina; Menziani, Maria Cristina; Fanelli, Francesca
abstract
Quantitative structure-activity relationship (QSAR) analysis has been done by making use of theoretical molecular descriptors on 13 non-congeneric alpha1a-adrenoceptor antagonists. Linear QSAR models have been obtained between ad hoc molecular shape and size descriptors, defined with respect to a reference ''super-molecule'', and the antagonistic potency. These results, obtained for a highly non-congeneric set of molecules, increase the potential of this approach and the probability of designing new leads. Finally, the reference supermolecule represents the best three-dimensional complementarity towards the alpha1a-adrenoceptor subtype being modelled by superimposing the two most active and selective alpha1a-antagonists.
1993
- Theoretical quantitative structure-activity analysis of quinuclidine-based muscarinic cholinergic receptor ligands
[Articolo su rivista]
Fanelli, Francesca; Menziani, Maria Cristina; Carotti, A; DE BENEDETTI, Pier Giuseppe
abstract
Quantitative structure-activity relationship analysis using ad hoc theoretical molecular descriptors was performed on a set of 22 quinuclidine-based muscarinic cholinergic receptor ligands. The results obtained support quantitatively a pharmacophoric interacting model which suggests that once the essential H-bonding interaction between the protonated quinuclidine nitrogen atom and a protophilic counterpart in the receptor is satisfied, different mechanisms of interaction become operative in order to differentiate between agonists, partial agonists and antagonists. In fact, the agonist behaviour is characterized by two H-bonding interactions, whereas mainly lipophilic interactions characterize the antagonistic behaviour. Finally, both the H-bond acceptor propensity of the agonists and lipophilic features of the antagonists are better accounted for by theoretical descriptors computed on the pharmacologically active protonated forms than by those computed on the neutral forms.
1993
- Three dimensional molecular modeling of the α1a-adrenoceptor. Direct 3D-QSAR modeling of selective antagonists
[Capitolo/Saggio]
Fanelli, Francesca; Menziani, Maria Cristina; Cocchi, Marina; DE BENEDETTI, Pier Giuseppe
abstract
three dimensional molecular modeling of the α1a-adrenoceptor. Direct 3D-QSAR modeling of selective antagonists
1992
- Molecular modeling and quantitaive structure activity relationship analysis using theoretical descriptors of 1,4-benzodioxan (WB-4101) related-compounds alpha-1-adrenergic antagonists
[Articolo su rivista]
P., Venturelli; Menziani, Maria Cristina; Cocchi, Marina; Fanelli, Francesca; DE BENEDETTI, Pier Giuseppe
abstract
Quantitative structure-activity relationship analysis using theoretical molecular descriptors was done on a set of 30 1,4-benzodioxan (WB-4101) related compounds which are alpha1-adrenoceptor antagonists. The results obtained confirm quantitatively and in terms of reactivity and molecular shape descriptors, the results of previous qualitative structure-activity relationship studies. It was found that the protonated amine function plays a crucial role in the potency of the alpha1-adrenoceptor antagonism due to a charge reinforced hydrogen bond with a primary nucleophilic site of the receptor. Furthermore, the more electrophilic (high SN1LUMO values) the NH2+ group, the stronger the charge reinforced hydrogen bond with the receptor and the higher the blocking activity. It was also found that the three-dimensional shape of the antagonists is more similar to the shape of the most active reference molecule (WB-4101) the more potent antagonists are. Finally, the reactivity (E(LUMO)) and the ad hoc shape (V(D)(norm)) descriptors were used to obtain a bilinear equation which accounts for about 77% of the total variance in the pharmacological data.
1991
- Conformational analysis, molecular modeling and quantitative structure-activity relationships studies of 2,4-diamino-6,7-dimethoxy-2-substituted quinazoline α1-adrenergic antagonists
[Articolo su rivista]
Rastelli, Giulio; Fanelli, Francesca; Menziani, Maria Cristina; Cocchi, Marina; DE BENEDETTI, Pier Giuseppe
abstract
Conformational analysis (AM1), modeling of the molecular shape (QUANTA 3.0) and quantitative structure-activity relationship analysis were done on a set of 16 2,4-diamino-6,7-dimethoxy-2-substituted quinazoline alpha-1-adrenoceptor antagonists (prazosin analogs). The results obtained show that the 2-substituents of the analogs considered are quite flexible. Furthermore, they suggest that, once the electronic requirements of the common quinazoline moiety are satisfied, the binding affinities are modulated by the molecular shape of the quinazoline 2-substituent, through the optimization of both dispersive and steric interactions and the hydrophobic contribution.