Activation and allosteric modulation of a muscarinic acetylcholine receptor

Abstract

Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.

Covalent agonists for studying G protein-coupled receptor activation

“…2B). The ability of dopamine analog 3 to activate WT D 2 R, as well as the ability of the covalent 3-D 2 R L94C complex to induce G protein-promoted signaling, was assessed in an inositol phosphate (IP) formation assay using HEK cells transiently transfected with a chimeric Gα qi5 protein and the WT and mutant receptor, respectively (6,23) (Fig. 2D and SI Appendix, Fig.…”

“…Although the crystallization of GPCR-G protein complexes is still notoriously difficult (4,26,27), a recent key strategy to obtain activestate structures comprised the use of conformationally selective nanobodies that serve as G protein mimetics and stabilize active conformations (5,6,24). Covalent ligand-receptor complexes can be useful for the generation of G protein-mimicking nanobodies because they prevent ligand dissociation during the immunization of llamas (28).…”

“…Purified WT β 2 AR or β 2 AR H93C reconstituted into recombinant HDL particles (22) was used as described (20) , and H 1 Y87C receptors was studied using IP accumulation assays as described (6,23). For activation studies with G i -coupled GPCRs, HEK 293 cells were transiently cotransfected with cDNA encoding for D 2 R L94C and the hybrid G protein Gα qi5 (Gα q protein with the last five amino acids at the C terminus replaced by the corresponding sequence of Gα i ; a gift from The J. David Gladstone Institutes) (36).…”

“…Low binding affinity with rapid association and dissociation rates leads to conformational heterogeneity that prevents the formation of diffraction-quality crystals. The rapid dissociation rate of agonists also makes it difficult to generate active-state stabilizing proteins, such as the camelid antibodies (nanobodies) that have been used to obtain active-state structures of the β 2 -adrenergic receptor (β 2 AR) (5) and M2 muscarinic receptor (6).…”

The deletion of M4 muscarinic receptors increases motor activity in females in the dark phase

“…Muscarinic receptors (MR) are typical members of G protein‐coupled receptors (Kruse et al., 2013) and can be divided into five subtypes (M 1 –M 5 ), which activate different G proteins (G q , G i ) (Eglen, 2012; Kow & Nathanson, 2012; Reiner & Nathanson, 2012). The often‐overlapping pattern of MR subtype expression and the lack of highly selective ligands toward a given MR subtype have precluded the precise delineation of MR subtype‐specific roles.…”