Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor

Abstract

Human muscarinic receptor, M 2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M 2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M 2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M 2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M 2 receptor compared to M 3 receptor leads to subtype selectivity of AF-DX 384.

Crystal structure of the M5 muscarinic acetylcholine receptor

“…Although introduction of the S117 3.39 R mutation resulted in a construct that binds the antagonists NMS or tiotropium with a slightly reduced affinity relative to the WT M 5 mAChR, the effect of the mutation on reducing ACh affinity was substantially more pronounced (Supplementary Figure 1b-e), consistent with the ability of the construct to favour an inactive over an active state. Similar differential effects on antagonist versus agonist affinity were previously observed for S 3.39 R at the M 2 mAChR (25). Notably, introduction of the S117 3.39 R mutation increased our M 5 mAChR yields during purification and resulted in crystals that diffracted to a resolution of 3.4 Å.…”

“…Nevertheless, it is still paradoxical that the addition of an allosteric modulator can clearly improve receptor crystallization and diffraction, though not be visible in any resulting structures. This phenomenon has been noted at other GPCRs, such as the M 2 mAChR that was crystallized in the presence of the modulator, alcuronium, and the CC chemokine receptor 2A that was crystallized in the presence of the modulator, AZD-6942, but where neither modulator could be observed in the resulting structures (25, 42).…”

“…(23) that predicted that mutation of the amino acid at position 3.39 (numbered according to Ballesteros-Weinstein (24)) to Arg would create a thermostabilized receptor by promoting an ionic bond between this residue and the highly conserved D 2.50 residue. Recently, the same S 3.39 R mutation was applied to the M 2 mAChR resulting in a series of higher resolution structures (25). Although introduction of the S117 3.39 R mutation resulted in a construct that binds the antagonists NMS or tiotropium with a slightly reduced affinity relative to the WT M 5 mAChR, the effect of the mutation on reducing ACh affinity was substantially more pronounced (Supplementary Figure 1b-e), consistent with the ability of the construct to favour an inactive over an active state.…”

“…This comparison demonstrated that the residues within the orthosteric pocket are absolutely conserved between the receptors. Although there is no tiotropium bound M 2 mAChR structure, there are now six different inactive state M 2 mAChR structures, which include structures bound with the non-selective ligands QNB and NMS, and the M 2 mAChR selective ligand, AF-DX384 (25). The 2.3 Å M 2 •NMS structure is most similar to the tiotropium bound mAChR structures, though residues Y 3.33 and Y 7.39 of the “tyrosine lid” (Y 3.33 , Y 6.51 , and Y 7.39 ) are positioned in a distinct conformation in comparison to the tiotropium bound structures.…”

“…In this study, we have determined a high-resolution crystal structure of the M 5 mAChR, thus allowing the first subtype-wide comparison for any aminergic GPCR subfamily. Introduction of the inactive state stabilizing mutation S117 3.39 R, which was recently used to stabilize the M 2 mAChR (25), was crucial to obtaining well-diffracting crystals and suggests that this mutation could be applied to aid the determination of inactive state structures for other related GPCRs. We further improved the resolution of the M 5 mAChR structure by adding allosteric modulators to the purified protein prior to crystallization.…”