Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as novel targets for allosteric drug design

Abstract

G protein-coupled receptors (GPCRs) are the most frequent targets of approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we utilized a combined computational and experimental framework that integrated extensive molecular dynamics simulations, Markov state models, and site-directed mutagenesis for investigating the conformational landscape of activation of the angiotensin II (AngII) type 1 receptor (AT1R), a prototypical class A GPCR. Our findings suggested a synergistic transition mechanism of AT1R activation. Importantly, a key intermediate state was found in the activation pathway. We discovered a novel “cryptic” binding site in the intracellular region of the receptor in the intermediate state. Furthermore, a bioluminescence resonance energy transfer analysis revealed the insensitivity of the endogenous AngII octapeptide agonist in the activation of the downstream Gq signaling and β-arrestin-mediated pathways, upon mutations of the predicted cryptic binding site, thereby suggesting an allosteric regulatory mechanism. Together, these findings not only provide a deeper understanding of AT1R activation at an atomic level, but also open potential avenues for the design of allosteric AT1R modulators. Consequently, this will provide a broad range of applications for GPCR biology, biophysics, and medicinal chemistry.