Molecular GPS: Receptor and G‐protein dynamics that drive selectivity in GPCRs

Abstract

Upon binding to agonists, G‐protein coupled receptors (GPCRs) couple selectively to specific G‐proteins (such as Gs, Gi, and Gq) or β‐arrestins to activate specific signaling pathways. Some GPCR agonists exhibit differential responses to signaling pathways, a phenomenon called “functional selectivity”. The mechanism by which the GPCR‐agonist pairing leads to G‐protein selectivity is relatively obscure. Structure based design of G‐protein selective ligands remains a challenge due to the paucity of information on the structural elements of the ligand‐GPCR‐G‐protein complexes that lead to G‐protein selectivity. We have used a combination of a multi‐resolution Molecular Dynamics (MD) method for enhanced conformational sampling of protein complexes, called the Generalized Newton‐Euler Inverse Mass Operator (GNEIMO) method and a genetically encoded FRET biosensor technique called Systematic Protein Affinity Strength Modulation (SPASM), to measure G‐protein specificity in live cells. The combination of GNEIMO and SPASM form a powerful, complementary and scalable toolset for studying the structural basis of G‐protein selectivity in several GPCR‐G‐protein pairings. SPASM sensors measure the interaction (through FRET) of two proteins tethered by a flexible α‐helical ER/K_linker; this tool permits measurement of non‐cognate GPCR‐G‐protein interactions which cannot be measured by traditional biochemical assays used for cell signaling. This allows for an unprecedented comparison of relative intrinsic affinities of different G‐proteins to a specific GPCR. In this study, we have generated the conformational ensemble for 7 class A GPCRs bound to a full agonist and a C‐terminal peptide from Gαs‐, Gαi‐, and Gαq‐proteins. We predicted the putative hotspot residues on the G‐protein that leads to selectivity and tested these mutations using SPASM for switching specificity between Gs, Gi and Gq receptors. We observe Gs peptide binding to a different location in the intracellular surface of GPCRs compared to Gi and Gq peptides. The homologous, anionic residues, E355 (Gq) and D350 (Gi), orient the G protein C‐terminus away from the Gs‐specific cavity in the GPCR interface. Gi binding is further distinguished from Gq at the conserved position of G352 (Gi) and N357 (Gq). The smaller G352 residue functions as a size‐filter for the smaller cavity of Gi‐signaling GPCRs. This work provides insight to molecular mechanisms governing G‐protein selectivity, and is imperative to the development of functionally selective ligands.Support or Funding InformationNIH R01 GM117923‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.