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

Shaoyong Lu,Zongtao Chai,Shuhua Zhou,Xinheng He,Jian Zhang,Jinpeng Sun,Ashfaq Ur Rehman,Zhao Yang,Junyan Wang,Jun Pu,Duan Ni

doi:10.1038/s41467-021-25020-9

Abstract

G protein-coupled receptors (GPCRs) are the most common proteins targeted by 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 apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT1 receptor) — a prototypical class A GPCR—activation. Our findings suggest a synergistic transition mechanism for AT1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Mutation of this cryptic site prevents activation of the downstream G protein signaling and β-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. Together, these findings provide a deeper understanding of AT1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.

Highlights

G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs
The GPCR-mediated signal transduction is always triggered by an extracellular signal to the orthosteric site located in the extracellular region of the 7TMs bundle center, which transduces the stimuli to the intracellular region, thereby leading to the engagement of the receptor with G proteins or β-arrestins[3,4]
Since the AT1 receptor has a constitutive activity for G proteins (Fig. 3C–E) and shows β-arrestin activity upon angiotensin II (AngII) binding or specific agonists[25], we explored the connection between different active states and biased signaling

Summary

Introduction

G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. Active, and inactive states of AT1 receptor exhibit marked structural divergences, it has been challenging to completely capture the large-scale conformational transitions along the activation pathway of the AT1 receptor experimentally It remains unclear how a dynamic pathway connects the inactive-to-active conformational transitions of the AT1 receptor, thereby hindering a deeper understanding of the comprehensive landscape of the activation mechanisms of this receptor as well as for other GPCRs. despite the availability of the inactive and active structures of the AT1 receptor, there are no allosteric modulators of this receptor reported to date, suggesting a challenge for targeting potential allosteric binding sites in the two available snapshots. In the best structurally and biochemically characterized GPCRs, the rhodopsin receptor, the activation pathway, and the corresponding intermediate states have been elucidated by NMR44, Fourier transform infrared spectroscopy[45], and MD simulations[46]

Methods

Results

Conclusion