Elucidation of calcium sensing receptor (CaSR) structural conformations using hydrogen-deuterium exchange mass spectroscopy

Although the number of experimentally elucidated G protein coupled receptor (GPCR) structures has increased in recent years, many questions regarding the structural basis of GPCR function and signalling remain unanswered. This is because GPCRs are highly dynamic proteins capable of adopting a multitude of distinct structural states that each facilitate macromolecular interactions critical for cellular signal transduction. In contrast, experimentally elucidated structures capture only a single conformation and therefore do not convey all conformational transitions essential for receptor signalling. The CaSR is ubiquitously expressed, and supports diverse biological processes through recognition of various endogenous ligands. The CaSR’s numerous roles may be attributed to its multi-liganded nature, with each ligand stabilising a distinct subset of CaSR structural states. To understand the conformational changes stabilised by different CaSR ligands, we investigated CaSR dynamics using amide hydrogen-deuterium exchange (HDX) mass spectroscopy. The rate of HDX depends on: the dynamics of a proteins secondary structure and the intrinsic chemical properties of the amino acid sequence. We measured agonist (calcium) driven activation of the CaSR extracellular domain (ECD) over time to identify agonist binding sites and conformational transitions in the absence and presence of L-tryptophan and phosphate. Our findings highlight regional plasticity of the CaSR ECD and identify distinct dynamic conformers in the presence of different ligands. Dynamic-function studies can elucidate new drug targets previously invisible to traditional structure-based drug design, broadening drug discovery efforts seeking to target the CaSR. The major finding of our study is the elucidation of distinct structural changes in the CaSR ECD in the presence of different endogenous CaSR ligands.