webPSN v2.0: a webserver to infer fingerprints of structural communication in biomacromolecules.

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.

Structural aspects of rod opsin and their implication in genetic diseases.

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.

Interconnecting Flexibility, Structural Communication, and Function in RhoGEF Oncoproteins.

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.

Dissecting intrinsic and ligand-induced structural communication in the ß3 headpiece of integrins.

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 ß3 domains from integrins αVß3 and αIIbß3 in their apo and ligand-bound states. RESULTS: Closed and open states of the ß headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/ß interfaces in the ßI 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 ß1/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 αßα-sandwich architecture of integrin ßI 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 ßI/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.

WebPSN: a web server for high-throughput investigation of structural communication in biomacromolecules.

UNLABELLED: 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.

Structure network analysis to gain insights into GPCR function.

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.

Catching Functional Modes and Structural Communication in Dbl Family Rho Guanine Nucleotide Exchange Factors.

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 α6 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 α6 and the linked PH domain as the portions of the tandem domain holding almost the totality of intrinsic and functional dynamics, with the α6/ß1 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.

Network and atomistic simulations unveil the structural determinants of mutations linked to retinal diseases.

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 PDEγ 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.

psnGPCRdb: The Structure-network Database of G Protein Coupled Receptors.

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.

Wordom update 2: A user-friendly program for the analysis of molecular structures and conformational ensembles.

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, ß-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.

Dimerization and ligand binding affect the structure network of A(2A) adenosine receptor.

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.

Conserved amino acids participate in the structure networks deputed to intramolecular communication in the lutropin receptor.

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.

Structural communication between the GTPase Sec4p and its activator Sec2p: Determinants of GEF activity and early deformations to nucleotide release.

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.

PSNtools for standalone and web-based structure network analyses of conformational ensembles.

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.

Wordom: a user-friendly program for the analysis of molecular structures, trajectories, and free energy surfaces.

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.

Structure network-based landscape of rhodopsin misfolding by mutations and algorithmic prediction of small chaperone action.

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.

Dynamics and structural communication in the ternary complex of fully phosphorylated V2 vasopressin receptor, vasopressin, and ß-arrestin 1.

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 ß-arrestin 1 (ß-arr1). Molecular simulations served to explore dynamics and structural communication in the ternary complex. Flexibility and mechanical profiles reflect fold of V2R and ß-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 ß-arr1. Two structurally and dynamically distinct receptor-arrestin interfaces assist the twist of the N- and C-terminal domains (ND and CD, respectively) of ß-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 ß-arr1 inter-domain twist angle correlates with the modes describing the essential subspace of the ternary complex. ß-arr1 motions are also influenced by the anchoring to the membrane of the C-edge-loops in the ß-arr1-CD. Overall fluctuations reveal a coupling between motions of the agonist binding site and of ß-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.

A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness.

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.

Structural Determinants of Constitutive Activation of Gα Proteins: Transducin as a Paradigm.

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, ß1-ß3, 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.

Network analysis to uncover the structural communication in GPCRs.

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 dynamics can be provided by atomistic molecular dynamics simulations or coarse-grained Elastic Network Models paired with Normal Mode Analysis (ENM-NMA). This chapter 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 PSN and the centrality of the retinal chromophore in differentiating the inactive and active states of the receptor.