Mass Spectrometry Coupled with Crosslinking as a Structural Tool for Structural Determination of Human α1 Glycine Receptor

The sensitivity, mass accuracy and robustness of mass spectrometry (MS) make it a valuable structural tool and when coupled with crosslinking, MS can complement structural determinations to refine and validate homology models. We have developed optimized and applied MS methods to identify crosslinked peptides in human α1 glycine receptor (GlyR). GlyRs are pentameric ligand-gated inhibitory receptors (pLGICs) that mediate neurotransmission in CNS. In these studies, A41C and H419C mutations are introduced in GlyR in a Cys null background and pure protein is obtained by baculovirus overexpression in insect cells. These sites were shown to be accessible in our previous studies. A41 is located nearby the pre-M1 and the M2-M3 loop flexible loop region that has been shown to be critical in gating and H419 is located in C-terminal tail of the receptor that is often truncated in structural studies conducted on other pLGICs (very little direct structural information being known in this region). Methane-thio-sulfonate (MTS)-benzophenone is used as a crosslinker to react with the cysteine in the sample and subsequent photo activation forms non-specific crosslinks that interrogate the local conformation around the original Cys site of attachment. Intra- and inter- subunit crosslinks are separated on a gel, trypsinized and subjected to multidimensional MS. Mass shifted peptides are further targeted for MS/MS and analysis of daughter ions identifies the specific sites of crosslinking. The large numbers of distance constraints identified were used to validate and refine our model of the resting state of human GlyR. These studies highlight the versatility of this general structural tool in providing complementary information regarding membrane protein structure that may help to refine and validate models, especially those regions that are highly flexible or absent in current structural models.