An extended structure of the intracellular domain of the Torpedo nicotinic acetylcholine receptor and its proposed interactions with rapsyn

Abstract

To gain insight into the interactions between rapsyn and the nAChR that induce clustering at the post-synaptic membrane, we refined a cryo-EM dataset using an intracellular domain focused strategy to obtain a 3.0 [A] map with the most extensive density yet for the intracellular domain of the Torpedo nAChR. The improved map allowed us to extend the structure beyond the MX -helix and prior to the MA -helix of the intracellular domain. The new structure defines a sharp N-terminal boundary of each MA -helix to place agrin-dependent phosphorylated tyrosines unambiguously within the flexible regions of the MX-MA loops. Two distinct conformations of the {delta} M4 -helix were also resolved, indicating that M4 conformational heterogeneity reflects intrinsic flexibility rather than a change in gating state. The new structural constraints defined for the MX-MA loop were then used to evaluate AlphaFold3-predicted full-length models of the nAChR, rapsyn, and various rapsyn-nAChR complexes, identifying a consistent, asymmetric 3:1 binding architecture where each rapsyn is always sandwiched between the MX-MA loops from two subunits and where each phospho-tyrosine is lodged in a cationic pocket formed by conserved residues implicated in congenital myasthenic syndromes. The defined architecture fits published cryo-ET maps of Torpedo post-synaptic membranes and explains how both phosphorylated tyrosines and myasthenic syndrome-causing rapsyn mutations modulate receptor clustering.