Site-specific Crosslinking Coupled with Mass Spectrometry as a Structural Tool in Studies of the Human α1 Glycine Receptor

Recent advances in mass spectrometry coupled with chemical crosslinking (CX-MS) can be applied for the structural interrogation of macromolecular complexes to identify statedependent distance constraints and provides a powerful complementary technique to other structural methods. In this study, we develop a CX-MS approach to identify the sites of crosslinking from a single targeted location within the human glycine receptor (GlyR) in a single apo/resting state. The GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neuronal transmission. A single cysteine residue was re-introduced into Cys null GlyR construct at position 41 within the extracellular domain of an overexpressed human a1 homomeric GlyR. After purification and reconstitution into vesicles, a methanethiosulfonate benzophenone heterobifunctional crosslinker was attached via formation of a disulfide bond, and the resting receptor is subsequently photocrosslinked. Monomeric and oligomeric bands from SDS-PAGE gels were then trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intra- and inter-subunit sites of crosslinking were differentiated and identified from single gel bands (pmols of purified GlyR), showing the utility of this approach to identify a diverse array of distance constraints of GlyR in its resting state. These studies highlight the potential of CX-MS as an experimental approach to identify state-dependent crosslinks of full length integral membrane protein assemblies in a native-like lipid environment.Competing Interest StatementThe authors have declared no competing interest.View Full Text