Structural studies of the human α₁ glycine receptor via site-specific chemical cross-linking coupled with mass spectrometry

By identifying distance constraints, chemical cross-linking coupled with mass spectrometry (CX-MS) can be a powerful complementary technique to other structural methods by interrogating macromolecular protein complexes under native-like conditions. In this study, we developed a CX-MS approach to identify the sites of chemical cross-linking from a single targeted location within the human α1 glycine receptor (α1 GlyR) in its apo state. The human α1 GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neurotransmission. A single chemically reactive cysteine was reintroduced into a Cys null α1 GlyR construct at position 41 within the extracellular domain of human α1 homomeric GlyR overexpressed in a baculoviral system. After purification and reconstitution into vesicles, methanethiosulfonate-benzophenone-alkyne, a heterotrifunctional cross-linker, was site specifically attached to Cys41 via disulfide bond formation. The resting receptor was then subjected to UV photocross-linking. Afterward, monomeric and oligomeric α1 GlyR bands from SDS-PAGE gels were trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intrasubunit and intersubunit sites of α1 GlyR cross-linking were differentiated and identified from single gel bands of purified protein, showing the utility of this experimental approach to identify a diverse array of distance constraints of the α1 GlyR in its resting state. These studies highlight CX-MS as an experimental approach to identify chemical cross-links within full-length integral membrane protein assemblies in a native-like lipid environment.