Landscape of G Protein-Coupled Receptor Function from Structure Networks

Ligand-Specified Signaling Efficacy Defined by Unique Transitions in G Protein Conformations

Decoding functional specialization in G protein-coupled receptors (GPCRs) through evolution-guided residue profiling.

BACKGROUND AND PURPOSE G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate physiological processes by enabling cells to detect and respond to diverse stimuli. Although many subfamily-specific functional hotspots have been described, the family-wide determinants of common and subfamily-specific functions remain incomplete. EXPERIMENTAL APPROACH Here, we developed an evolutionary framework utilizing conservation within orthologs and variation across paralogs to classify positions as common residues (CRs) or selective residues (SRs). KEY RESULTS Common residues (CRs) cluster in sites linked to structural stability and activation, whereas SRs concentrate at selective interfaces involved in ligand and transducer binding. SR distributions across families revealed that some classes mainly diversify through changes in ligand-recognition sites, whereas others through changes at transducer-binding interfaces. We also uncovered CRs involved in family-specific and cross-family motifs, including conserved disulfide bridges and cholesterol-contact sites. CONCLUSIONS AND IMPLICATIONS Together, these findings provide an evolutionary blueprint of family-wide features, reinforce known associations and deliver testable hypotheses that are especially valuable for understudied families.

Tools to investigate class B1 GPCR conformational landscapes with cryo-EM and beyond.

G protein-coupled receptors (GPCRs) are membrane proteins that act as signal transducers across cell membranes. Class B1 GPCRs, a subset of 15 receptors activated by peptide hormones, are involved in important physiological processes and diseases, making them a popular target for drug development. GPCRs are dynamic proteins and can adopt a myriad of conformational states, allowing them to bind and activate multiple intracellular signal transducers, including G proteins and β-arrestins, though all class B1 GPCRs primarily couple to the stimulatory G protein (Gs). Cryogenic electron microscopy (cryo-EM) structures of all class B1 GPCRs bound to Gs are available and provide meaningful insights into receptor function. However, there is a dearth of structural information on class B1 GPCRs in inactive and intermediate states or bound to other signal transducers, meaning we are currently only afforded a small vista into the conformational landscape these GPCRs sample. As cryo-EM-based 3D reconstructions are heavily dependent on protein stability and conformational homogeneity, currently available structures are largely limited to only those most stable conformations (i.e. Gs complexes). The present review focuses on technical aspects of obtaining class B1 GPCR structures using cryo-EM and new in silico methods that allow insight into unseen GPCR conformations, revealing more structural details of the conformational landscape.

Residue Interaction Network Reveals Allosteric Pathways Linking Orthosteric and Intracellular Sites in Class A GPCRs

Understanding how allosteric modulators influence protein dynamics is essential for guiding drug design. This work analyses a total of 45 μs of classical molecular dynamics simulations for four class A G-protein-coupled receptors (GPCRs), namely the Complement C5a receptor (C5AR1), the Purinergic Receptor P2Y (P2RY1), and the Cannabinoid Receptors 1 and 2 (CNR1/CNR2). Protein dynamics is essential to detect the shallow extrahelical binding sites, such as the one found in P2RY1. Current methods for computing Allosteric Communication Networks (ACNs) produce complex outputs requiring expert interpretation. To address this, we focus on the shortest paths of information transfer between the orthosteric and G-protein binding sites in Class A GPCRs. Our retrospective analysis reveals state- and bias ligand-dependent residue interactions along these communication pathways. Furthermore, focusing on the predicted binding site of allosteric modulator EC21a at cannabinoid receptors, the ACN framework was used to prioritize two residues for mutational analysis that may contribute to allosteric communication.

Mapping the relationship between receptor dynamics and function in G protein-coupled receptors using molecular dynamics.

G protein-coupled receptors (GPCRs) are key regulators of cellular signaling and major drug targets. Although X-ray crystallography and cryo-electron microscopy have provided high-resolution receptor structures, these static snapshots capture only a fraction of the conformational states underlying GPCR function. Molecular dynamics (MD) simulations complement experimental structures by enabling the mapping of receptor conformational landscapes and their relationship to functional outcomes. Large-scale simulation resources such as GPCRmd allow systematic exploration and comparison of GPCR motions across receptors and ligands. MD studies reveal transient conformational states, cryptic binding pockets, lipid- and water-mediated interactions, and allosteric communication networks that shape ligand recognition and the impact of signaling bias.

Benchmarking Docking Protocols for GPCR Allosteric Modulators

GaMD ensemble docking improved early AM enrichment across all four targets under at least one program, and the Boltz-2 deep-learning program showed minimal sensitivity to GaMD templates and underperformed conventional docking, suggesting its affinity predictions complement rather than replace physics- and empirical-based docking approaches for GPCR AM screening.