Fig. 2: Conserved Hs-M1 binding site residues determine the selective affinity of Ir-mAChR-A and -B for atropine.
a Structural alignment of the resolved Hs-M1 (grey), and predicted Ir-mAChR-A (black) and -B (cyan) structures showing atropine (spheres: red, oxygen; blue, nitrogen; green, carbon) and key binding residues (sticks). Upper inset: sequence alignment of Hs-M1 atropine-binding residues with Ir-mAChR-A and -B (asterisk, identical; colon, strongly similar; period, weakly similar). Lower inset: Hs-M1 atropine-binding residues (grey) indicating substitutions between Ir-mAChR-A/Hs-M1 and -B (black). b A bird’s-eye view (90° rotation of a) of superimposed Hs-M1, Ir-mAChR-A and -B showing residues coordinating atropine in the binding site. c Sequence alignment of human M1–M5 receptors and tick mAChR-A and -B highlighting residues in TM3, TM5, and TM6 that determine atropine selectivity. Species: Ir, I. ricinus; Is, I. scapularis; Rm, Rhipicephalus microplus; Rs, Rhipicephalus sanguineus; Da, Dermacentor andersoni; Ds, Dermacentor silvarum. Full sequences are provided in Supplementary Dataset 1. d 500-ns molecular dynamics trajectories showing RMSD of atropine (y-axis) across single Hs-M1, reflecting Ir-mAChR-B substitutions, and the Hs-M1Ir4B (e). WT, wild type. f Fragments of Ir-mAChR-A and -B sequences showing mutation sites (green and yellow, respectively) used to generate Ir-mAChR-A4B and -B4A mutant variants. g, h Aequorin assays comparing responses of WT and Ir-mAChR-A4B and -B4A to 45 μM agonists; ACh responses were normalised to (100%) in each of the three biological replicates. Effect of atropine pre-treatment on 30 μM muscarine- (i) and arecoline- (j) induced responses of WT and Ir-mAChR-A4B or -B4A in CHO/Gα15(16) aequorin assay. (g–j) Data are from three independent transfections (N = 3). Statistics: (i, j) nonlinear regression. Data are mean ± SD. Source data are available in the Source Data file.
