Correcting congenital myasthenia-associated acetylcholine receptor defects

a, Representative whole-cell electrophysiology of EC-216 potentiation on wild-type muscle receptor. b, Cryo-EM density map of εP121L mutant bound to XG-590 at the ε binding site. XG-590 density was shown. The XG-590 density at the β site was stronger than the ε site, here shown at a lower threshold than that in Fig. 1. c, Interactions to stabilize XG-590 binding in the ε binding site. Black dashed lines indicate the polar interactions. d, e, Plots of two XG-590 binding sites show the interactions. Red dashes, hydrophobic interaction; gray arrow, backbone polar interaction; black arrow, side-chain polar interaction. f, Structural comparisons of two binding sites of XG-590 on the εP121L mutant receptor. The β and ε subunits are superimposed to illustrate the similarities and differences between the two XG-590 binding sites. XG-590 inserts more deeply at the β subunit site due to the larger cavity compared with the ε subunit site. In the ε subunit, F454 would sterically clash with XG-590 positioned as in the β site, as they partially occupy the same cavity volume. g, Comparison of potentiating effect between wild type receptor and the triple mutant in the 2 µM ACh and 0.6 µM XG-590. Data are represented as mean ± s.e.m. of biological replicates (n value is labeled in each bar graph). h, Comparison of potentiating effect between wild type receptor and the quadruple mutant in the 2 µM ACh and different concentrations of XG-590. The unpaired two-tailed t-test was used; data are represented as mean ± s.e.m. of biological replicates (n value is labeled in each bar within the graph). In panels g and h, no substantial reduction was observed in our mutagenesis analysis, which may reflect a non-canonical binding mode in which XG-590 interacts within the transmembrane domain through extensive hydrophobic contacts in a dynamic lipid environment (Extended Data Fig. 1d,e,j), thereby rendering the modulation relatively resistant to few point mutations. We cannot exclude alternative possibilities, including XG-590 binding to a conformation-specific pocket in the activated state that may differ from that captured in our structures, or engagement of an additional weak binding site at the equivalent position in the δ subunit, as suggested by much weaker density features in our maps (Extended Data Fig. 1i), which could partially compensate for perturbations at primary sites. i, Discontinuous densities observed at the equivalent position in the δ subunit. j, Extensive lipid densities are observed adjacent to XG-590 at both sites. k, Chemical structures of meloxicam (top panel), which is the head part of two PAMs and a modified compound AS3580239 (bottom panel34) from XG-590 and EC-216. Red dashed circle indicates the modified region. l, Modeled structure of AS3580239 based on the XG-590-bound εP121L structure suggests more interactions result from the modification.