Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.
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We thank L. Lopez for generating initial M4 homology models. This work was funded by Program Grant APP1055134 of the National Health and Medical Research Council (NHMRC) of Australia (A.C., P.M.S.). Portions of this work were supported by a Lilly Research Award Program grant. W.I.W. and B.K.K. were supported by the Mathers Foundation. A.C. is a Senior Principal, and P.M.S. a Principal, Research Fellow of the NHMRC. GM/CA @ APS has been funded in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-GM-1104). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract number W-31-109-ENG-38.
C.C.F., M.G.B. and D.E. are employees of Eli Lilly.
Extended data figures and tables
a, b, Crystallization constructs used for the (a) M1 receptor and (b) M4 receptor. All constructs contain an N-terminal Flag epitope (yellow), C-terminal histidine tag (purple), and a T4L lysozyme fusion protein (red). For the M4 receptor, initial constructs diffracted out to 4 Å; however, the diffraction data appeared to suffer from a lattice translocation disorder and were unsolvable. The final crystallization construct contained a shortened N terminus with an HRV 3C cleavage site, shown in dark green, and a minimal T4 lysozyme fusion (mT4L)26, shown in red. c, Snake-plot diagram of the best diffracting M4 mAChR construct coloured according to a. Residues coloured blue are single-point mutations from this study, and residues coloured orange are previously studied mutations20,21. d, Size-exclusion chromatography trace of purified monodispersed M4-mT4L bound to tiotropium. e, Crystals of M4-mT4L obtained in lipidic cubic phase and observed under circularly polarized light.
a–c, The sequence alignment of the human M1–M5 receptors (d) was determined on the ConSurf server to calculate amino-acid conservation scores60,61. Conservation scores for each residue were mapped62,63 onto the M4 structure and coloured as a gradient from blue (highly conserved) to red (least conserved) with views from the (b) extracellular and (c) intracellular sides. The radius of the cartoon increases as the residues at each position become more poorly conserved. Tiotropium and PEG 300 from the M4 structure are shown as spheres and coloured with carbon in white, oxygen in red, nitrogen in blue, and sulfur in yellow. d, Amino-acid sequences of the human M1–M5 receptors were aligned using the ClustalW2 server64. Alpha helical regions are shown as blue boxes as determined by the consensus of the M1–M4 structures. The most conserved residue in each TM (X.50) is in bold lettering. Regions of the N terminus, C terminus, and ICL3 regions are removed for space and clarity. Insertion points of the T4 lysozyme fusion proteins between TM5 and TM6 are underlined with bold lettering. Residues from the orthosteric binding-site are highlighted in red and allosteric binding-site residues in blue. Residues that contribute to both sites are coloured in yellow.
The receptors shown are aligned and coloured as in Fig. 1. a, b, The M1 receptor was crystallized with the Flag peptide (DYKDDDD; coloured cyan sticks) co-bound on the cytoplasmic surface. Residues of the M1 receptor within 4 Å of the Flag peptide are shown as magenta coloured sticks with views from the (a) membrane and (b) cytoplasmic side. c, The linkage between TM7 and helix 8 of the M1 receptor undergoes a bend starting with a change in rotamer of residue Y7.53, which may be a result of perturbations in TM6 due to the Flag peptide. d, The M1-N110Q3.37 mutation causes a slight bulge in TM4 due to the loss of a hydrogen bond with S4.53. e, Chain B of the M4 receptor has an intact ionic lock with R3.50 forming hydrogen bonds with T6.34 and E6.30.
The receptors shown are aligned and coloured as in Fig. 1. a, Superposition of the poses of pirenzepine from the IFD experiments. b, Comparison of the pirenzepine poses for the M1 and M4 receptor with residues that contribute to the orthosteric site of the M1–M4 receptors (several residues omitted for clarity).
a, The cross section of the solvent accessible surface area of the M4 receptor is coloured blue. Tiotropium and PEG 300 are shown as spheres with respective carbons coloured white and peach. The aromatic cage of covering tiotropium is highlighted in orange b, View from the extracellular side with residues that contact PEG 300 shown as spheres. c, Dissociation kinetics of [3H]NMS in the presence of PEG 300. [3H]NMS was incubated with M4-mT4L membranes at 37 °C for 3 h, followed by addition of 10 μM atropine ± PEG 300 at the indicated concentrations and time points. Representative data from three experiments, performed in duplicate, fitted to a one-phase exponential decay are shown. d, PEG 300 has an apparent binding affinity for the NMS-occupied receptor of approximately 10 mM (log(IC50) = −1.95 ± 0.02).
a, b, The molecular interactions between the (a) orthosteric and (b) allosteric binding sites are shown by the program MOE65 for the inactive (M4•tiotropium structure) and active states (M4•acetylcholine•LY2033298 model). Residues with a bold outline were selected in this study or others20,21 as single-point mutations.
Extended Data Figure 7 Identification of key residues that govern LY2033298 affinity and binding cooperativity with ACh at the M4 receptor.
Competition between a fixed concentration of [3H]QNB and increasing concentrations of ACh (black circles), LY2033298 (blue triangles), or LY2033298 in the presence of an IC20 concentration of ACh (red squares) are shown. The curves drawn through the points represent the best global fit of an extended ternary complex model. For data sets where the binding of [3H]QNB changed by less than 10% at 10−5M LY2033298 relative to zero LY2033298, the value of α′ was fixed to 1 (connecting line shown). Data points represent the mean ± s.e.m. of at least three experiments performed in triplicate.
Extended Data Figure 8 Comparison of cooperativity network residues between the inactive and active-states.
a, b, Chemical structures of (a) the M4 ligands used in this study and (b) the M2 ligands from the active-state crystal structures (PDB accession number 4MQT and 4MQS). c–f, Mapping of the allosteric network onto the (c, d) inactive M4 structure (blue residues), M4 active-state model (orange residues) and (e, f) the inactive (yellow residues) and active-state M2 structures (magenta and green residues) with views from the (c, e) membrane or (d, f) extracellular surface. Ligands are coloured according to element: carbon, cyan; oxygen, red; nitrogen, blue; sulfur, yellow; chlorine, green.
Comparison of active-state M1 (green) and M4 (orange) models bound to LY2033298 and acetylcholine, with acetylcholine and LY2033298 shown as sticks and coloured according to element: carbon, white; oxygen, red; nitrogen, blue; sulfur, yellow; chlorine, green. Several residues surrounding LY2033298 are shown as sticks and coloured according to receptor. M4-N4236.58 is predicted to undergo significant movement between the inactive and active states to form a hydrogen bond with the methoxy group of LY2033298. In the M1 receptor this residue is a serine (S3886.58) and is unable to form a similar hydrogen bond. However, mutation of N4236.58 to alanine at the M4 receptor results in no loss of LY2033298 affinity, but does result in a sixfold loss in cooperativity between acetylcholine and LY2033298 (Supplementary Table 3). This is suggestive of selectivity being derived through cooperativity as a possible mechanism between the M1 and M4 receptors. Additional determinants for M1 and M4 selectivity could also arise through differences in residues on TMs 2 and 7, which contribute to (I932.65) or sit proximal to (D4327.32 and S4367.36) the allosteric network.
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Thal, D., Sun, B., Feng, D. et al. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531, 335–340 (2016). https://doi.org/10.1038/nature17188
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