Angelo Felline

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.

Dimerization and ligand binding affect the structure network of A2A 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.

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

Graph theory is being increasingly used to study the structural communication in biomolecular systems. This requires incorporating information on the systems 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.

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.

Light on the structural communication in Ras GTPases

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, interfunctional states and interfamily 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.

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 systems 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.

Quaternary Structure Predictions and Structural Communication Features of GPCR Dimers

In spite of the ever-increasing evidence that G protein-coupled receptors (GPCRs) form dimers/oligomers, the biological role(s) and structural architecture of homologous and heterologous receptor aggregation 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.

Computational Screening of Rhodopsin Mutations Associated with Retinitis Pigmentosa.

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 β-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 β-hairpin, proving the effectiveness of the implicit solvent model in reproducing the free energy landscape of β-hairpin formation. Eight out of the 40 EL2 mutants resulted in misfolding effects on the native β-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 β-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.

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.