Structural and Dynamic Mechanisms of Allostery at the M4 Muscarinic Acetylcholine Receptor

Abstract

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular level understanding of general principals governing the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M₄ muscarinic acetylcholine receptor (M₄ mAChR) is a well-validated and clinically relevant allosteric drug target. Here, we present high-resolution cryo-electron microscopy structures of the M₄ mAChR bound to a cognate G_i1 protein and the high-affinity agonist, iperoxo, in the absence and presence of two different positive allosteric modulators, LY2033298 or VU0467154. We also determined the structure of the M₄ mAChR-G_i1 complex bound to its endogenous agonist, ACh. Structural comparisons, together with molecular dynamics, mutagenesis and pharmacological characterization, provided unprecedented insights into the role of structure and dynamics in orthosteric and allosteric ligand binding, global mechanisms of receptor activation, cooperativity, probe-dependence, and species variability; key phenomena underpinning contemporary GPCR drug discovery.

Keywords: G protein coupled-receptor, M4 muscarinic acetylcholine receptor, allostery, cryoelectron microscopy, molecular dynamics, pharmacology, drug discovery