Human Α1 Glycine Receptor Allostery As Identified by State-Dependent Crosslinking Studies

In order to better understand the allostery at a molecular level, state-dependent crosslinking studies coupled with multidimensional MS were conducted on glycine receptor (GlyR) stabilized in different allosteric states. Predominant allosteric states were stabilized using wild type or mutated receptor in the presence of selected ligands: resting (no ligand), desensitized (saturating glycine) and open (non-desensitized ivermectin (IVM) gated F207A/A288G GlyR). Recent advances in technology have led to numerous notable structures of membrane proteins. While they provide valuable information about the structure of membrane proteins they often require truncations of flexible regions often focus on bacterial proteins or homologs from other species given the need for heterologous expression and provide static images with potentially limited dynamics. A developed photo crosslinking methodology linked with mass spectrometric analysis on systematically generated single Cys mutations in GlyR with both Cys null and IVM sensitive backgrounds to enable us to study state-dependent structure of GlyR in comparative crosslinking studies. Studies were conducted on A41C and H419C mutants. A41 is shown to be in proximity to the pre-M1 and the M2-M3 loop region crucial for gating and very little or no information on H419 is available as it located in C-terminal tail of the receptor that is often truncated in structural studies conducted on other pLGICs. Distance constraints obtained from the above studies for the different states of GlyR identified specific crosslinks unique to each conformational state and identified potential motions in the receptor upon gating and desensitization. The defined constraints were used to update our model of human α1 GlyR and provide insight into channel function. Significantly, this methodological approach is amenable to study any allosteric protein and complement other high resolution structural studies in identifying protein dynamics.