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bioRxiv · 10.1101/2020.06.29.178681

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

Veeramachaneni, R. J.·Donelan, C. A.·Tomcho, K. A.·Aggarwal, S.·Lapinsky, D. J.·Cascio, M.
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Abstract

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

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Veeramachaneni, R. J., Donelan, C. A., Tomcho, K. A., Aggarwal, S., Lapinsky, D. J., Cascio, M.. 2020-06-30. Site-specific Crosslinking Coupled with Mass Spectrometry as a Structural Tool in Studies of the Human α1 Glycine Receptor. https://doi.org/10.1101/2020.06.29.178681

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