Francesca Fanelli

G protein-coupled receptors (GPCRs) represent the therapeutic targets for an estimated 30–40% of marketed drugs. By translating the majority of the GPCR structures from the Protein Data Bank into structure graphs and analyzing over 2 million structural contacts, this study maps the landscape of GPCR classification and function. A minimal subset of just 30 specific contacts is sufficient to define the signatures of distinct receptor classes, subfamilies, types, subtypes, and functional states. These structural signatures successfully assigned classifications to the orphan GPCRs. Upon activation, class A GPCRs undergo the most radical network reorganization of all classes, retaining 0% of their state-specific contacts when transitioning from the inactive to the active state, a stark contrast to the 32–50% retention observed in classes B1, C, and F. Despite this complete contact turnover across the 7TM bundle, class A activation remains anchored by a highly conserved core of ten universal network nodes. Analysis of representative shortest communication pathways (metapaths) demonstrates that class A GPCRs rely on these highly conserved nodes to bridge structural communication between the orthosteric ligand-binding pocket and the intracellular G-protein-binding site, regardless of the functional state. Furthermore, these metapaths intersect directly with known allosteric binding sites for small allosteric modulators. G protein binding structurally reorganizes the receptor network, funneling a multitude of potential communication pathways into a few preferential routes. These pathways culminate at the highly conserved arginine residue of the E/DRY motif that acts as a key mediator of G-protein recognition, while structural divergences at the receptor-G protein interface dictate the distinctive pathways of specific G-protein signaling. The wide analysis was able to capture key aspects of the structural communication within the GPCR superfamily with implications in drug discovery.

The G-protein coupled receptor (GPCR) superfamily directs central roles in many physiological and pathophysiological processes via diverse and complex mechanisms. GPCRs can exhibit signal pleiotropy via formation of di/oligomers both with themselves and other GPCRs. A deeper understanding of the molecular basis and functional role of oligomerization would facilitate rational design of activity-selective ligands. A structural model of the D2 dopamine receptor (D ₂ R) homomer identified distinct combinations of substitutions likely to stabilize protomer interactions. Molecular modelling of β-arrestin-2 (βarr2) bound to predicted dimer models suggests a 2:2 receptor:βarr2 stoichiometry, with the dimer favouring βarr2 over Gαi coupling. A combination of biochemical, biophysical and super-resolution, single molecule imaging approaches demonstrated that the D ₂ R mutant homomers exhibited greater stability. The mutant D ₂ R homomers also exhibited bias towards recruitment of the GPCR adaptor protein βarr2 with either faster or ligand-independent βarr2 recruitment, increased internalization and reprogrammed regulation of ERK signaling. Through GPCR dimer-stabilization, we propose that D ₂ R di/oligomerization has a role in βarr2-biased signaling.

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).

Structure graphs, in which interacting amino acids/nucleotides correspond to linked nodes, represent cutting-edge tools to investigate macromolecular function.

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.

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.

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.