Molecular determinants of ligand efficacy and potency in GPCR signaling

INTRODUCTION Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) bind extracellular ligands to modulate intracellular signaling responses. Two fundamental properties of ligand-receptor-signaling systems are efficacy (the maximum achievable response) and potency (the ligand concentration required to produce the half-maximal response). Although efficacy and potency have been measured for numerous ligand-receptor-signaling systems for several decades, the molecular determinants and principles governing these pharmacological properties remain a mystery.RATIONALE Understanding how each receptor residue contributes to efficacy and potency can aid in designing drugs to elicit desired signaling responses. Using the adrenaline-beta 2 adrenergic receptor (beta 2AR)-G alpha s system, we perturbed the side chain of each of the receptor's 412 residues and determined the impact on efficacy and potency. By developing a data science framework that integrates pharmacological and structural data, we contextualized ligand-induced structural changes and revealed the principles of efficacy and potency.RESULTS Only 20% of the beta 2AR residues contribute to the receptor's pharmacological properties. One-third of those pharmacologically relevant residues map to ligand- or G protein-binding sites or to evolutionarily conserved motifs; the other two-thirds are distributed throughout the receptor. All ligand-binding residues are important for signaling but show differential contributions to efficacy and potency, which indicates that specific contacts between ligand and receptor could be modulated to fine-tune pharmacological properties. By contrast, only one-third of the receptor residues that contact the G protein contribute to pharmacology, which indicates that these positions tolerate mutations and provides an explanation for the evolvability of G protein selectivity. We integrated structural data about active and inactive receptor conformations with pharmacological measurements. Not all residues that undergo structural change are pharmacologically important, and several pharmacologically important residues do not undergo structural change upon activation. We categorized all receptor residues into four classes: Driver and passenger residues mediate active state-specific contacts, and, whereas drivers affect pharmacology when mutated, passengers do not. Modulator and bystander residues do not mediate active state-specific contacts, but modulators affect pharmacology when mutated, whereas bystanders do not. We uncovered an allosteric network of active state-specific contacts mediated by driver residues from the ligand-binding pocket to the G protein-binding interface, thereby identifying structural changes that are pharmacologically relevant. Modulator residues are located near the allosteric network and functional sites. Surface-exposed driver, modulator, and passenger residues represent key allosteric sites and potential targets for the development of new allosteric ligands. Analysis of human polymorphisms and the conservation of residues across species revealed that passenger, modulator, and driver residues are under increasing selection pressure compared with bystander residues in this receptor.RESULTS Only 20% of the beta 2AR residues contribute to the receptor's pharmacological properties. One-third of those pharmacologically relevant residues map to ligand- or G protein-binding sites or to evolutionarily conserved motifs; the other two-thirds are distributed throughout the receptor. All ligand-binding residues are important for signaling but show differential contributions to efficacy and potency, which indicates that specific contacts between ligand and receptor could be modulated to fine-tune pharmacological properties. By contrast, only one-third of the receptor residues that contact the G protein contribute to pharmacology, which indicates that these positions tolerate mutations and provides an explanation for the evolvability of G protein selectivity. We integrated structural data about active and inactive receptor conformations with pharmacological measurements. Not all residues that undergo structural change are pharmacologically important, and several pharmacologically important residues do not undergo structural change upon activation. We categorized all receptor residues into four classes: Driver and passenger residues mediate active state-specific contacts, and, whereas drivers affect pharmacology when mutated, passengers do not. Modulator and bystander residues do not mediate active state-specific contacts, but modulators affect pharmacology when mutated, whereas bystanders do not. We uncovered an allosteric network of active state-specific contacts mediated by driver residues from the ligand-binding pocket to the G protein-binding interface, thereby identifying structural changes that are pharmacologically relevant. Modulator residues are located near the allosteric network and functional sites. Surface-exposed driver, modulator, and passenger residues represent key allosteric sites and potential targets for the development of new allosteric ligands. Analysis of human polymorphisms and the conservation of residues across species revealed that passenger, modulator, and driver residues are under increasing selection pressure compared with bystander residues in this receptor.RESULTS Only 20% of the beta 2AR residues contribute to the receptor's pharmacological properties. One-third of those pharmacologically relevant residues map to ligand- or G protein-binding sites or to evolutionarily conserved motifs; the other two-thirds are distributed throughout the receptor. All ligand-binding residues are important for signaling but show differential contributions to efficacy and potency, which indicates that specific contacts between ligand and receptor could be modulated to fine-tune pharmacological properties. By contrast, only one-third of the receptor residues that contact the G protein contribute to pharmacology, which indicates that these positions tolerate mutations and provides an explanation for the evolvability of G protein selectivity. We integrated structural data about active and inactive receptor conformations with pharmacological measurements. Not all residues that undergo structural change are pharmacologically important, and several pharmacologically important residues do not undergo structural change upon activation. We categorized all receptor residues into four classes: Driver and passenger residues mediate active state-specific contacts, and, whereas drivers affect pharmacology when mutated, passengers do not. Modulator and bystander residues do not mediate active state-specific contacts, but modulators affect pharmacology when mutated, whereas bystanders do not. We uncovered an allosteric network of active state-specific contacts mediated by driver residues from the ligand-binding pocket to the G protein-binding interface, thereby identifying structural changes that are pharmacologically relevant. Modulator residues are located near the allosteric network and functional sites. Surface-exposed driver, modulator, and passenger residues represent key allosteric sites and potential targets for the development of new allosteric ligands. Analysis of human polymorphisms and the conservation of residues across species revealed that passenger, modulator, and driver residues are under increasing selection pressure compared with bystander residues in this receptor.CONCLUSION Our work reveals how a GPCR decodes and translates the information encoded in a ligand to mediate a distinct signaling response. We anticipate that the application of this data science framework will enable the design of orthosteric and allosteric molecules that can elicit defined signaling responses. Determinants and molecular origins of efficacy and potency in a ligand-receptor-signaling system. (A and B) By developing a data science framework that integrates experimental values of adrenaline-stimulated Gs signaling for 412 mutants of the beta 2AR (A) and data on structural changes upon ligand-induced receptor activation (B), we reveal the determinants and the allosteric network governing ligand efficacy and potency in this prototypical GPCR system.