Crosslinking-Mass Spectrometry of Targeted Single Cys Mutants to Refine Allostery and Model Building in the Glycine Receptor

Abstract

Studies regarding glycine receptor (GlyR) allostery can help provide insight into receptor function and allow us to develop novel therapeutics to modulate its activity. To conduct these studies, single active thiols are introduced into a Cys null background (C41S/C290A/C345S) or in the same background with a double mutation (F207G/A288G) that causes GlyR to be activated in a non-desensitizing manner to ivermectin (IVM). The crosslinker, MTS-Benzophenone, is attached via disulfide linkage to a single active Cys and the receptor is enriched in an allosteric state, resting (no ligand), desensitized (excess glycine) or open (F207G/A288G + ivermectin), before photoactivation and generation of non-specific crosslinks that examine the local topography near the sites of attachment. The crosslinked peptides, generated by trypsin digestion of monomeric and oligomeric bands, are analyzed using crosslinking mass spectrometry (CX-MS) and tandem mass spectrometry (MSMS) to sensitively identify sites of intra- and intermolecular crosslinking, respectively. In this study, we examine the network of interactions generated by three mutations, introduced singly at A41C, H419C or M287C in the resting, open and desensitized states. Recent studies using M287C, a position near the top of TM3, potentially provides information on how this helix moves and how the membrane regions are involved in the opening and closing of the channel.