Targeted State Dependent Crosslinking Mass Spectrometry (CXMS) of the Human Alpha 1 Glycine Receptor (Glyr)

Site-specific single Cys-mutations coupled with crosslinking mass spectrometry (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. Single, active thiols are 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 to be activated by ivermectin (IVM). State-dependent crosslinking with methanethiosulfonate benzophenone to a single thiol of purified, vesicle reconstituted GlyR will occur via photoactivation after enrichment of the receptor in different allosteric states: resting (no ligand), open (F207G/A288G + IVM), or desensitized (excess glycine). Digested peptides are analyzed using liquid chromatography mass spectrometry to identify sites of intra- and intermolecular crosslinking. Tandem MS of mass-shifted precursor ions further refine these distance constraints. The network of interactions from independent studies targeting different single Cys GlyR provides evidence of allosteric changes between the three states, as well as direct topological information of regions of the receptor unresolved in other high resolution structures of pLGICs.