Using a Network of Single Site Specific Mutations and Crosslinking Mass Spectrometry (CXMS) to Refine the Structure and Dynamics of the Human Alpha 1 Glycine Receptor (GLYR)

Abstract

A network of site-specific single Cys-mutations coupled with CX-MS can be used to elucidate a more refined structure of GlyR and obtain a more definitive understanding of pentameric ligand-gated ion channel (pLGIC) allostery. Each Cys-mutant is introduced into an α1 homomeric Cys null background (C41S/C290A/C345S), or in the same background with F207G/A288G mutation that allows non-desensitizing GlyR activation by ivermectin (IVM). State-dependent crosslinking with methanethiosulfonate benzophenone to a single thiol of purified, vesicle reconstituted GlyR are conducted after enriching the receptor in different allosteric states: resting (no ligand), open (F207G/A288G + IVM), or desensitized (excess glycine). Digested peptides are analyzed via liquid chromatography mass spectrometry to identify sites of intra- and intermolecular crosslinking. Tandem MS of mass-shifted precursor ions further refine these distance constraints. Independent comparative studies targeting different single Cys GlyR (M287C, K116C, K206C) provides evidence of allosteric changes between the three states, as well as direct topological information of unresolved regions, most notably the M3-M4 loop, in other high-resolution structures of pLGICs.