Investigation of GPCR-G protein coupling by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) : the GPBAR1 case.

ABSTRACT: G protein-coupled receptors (GPCRs) are key classes of proteins for drug discovery research as many of them are still orphans and their function poorly known. Their activation mechanism is roughly divided in two states : active and unactive states however related dynamics to switch from one state to another are poorly understood. The rise of cryogenic electron microscopy (cryo-EM) allowed to determine numerous GPCR high resolution structures in complex with G-proteins or not during the last couple of years. In this structural biology landscape, a few alternative techniques such as Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) have emerged as particularly suitable to characterize protein dynamics including membrane proteins. Here, for the first time, we optimized an HDX-MS workflow to characterize a class A GPCR, the G-protein-coupled bile acid receptor (also known as Gpbar1 or TGR5), in complex with G-proteins and a stabilizing nanobody. We, first, extensively describe the HDX-MS method optimization and related useful information we got regarding folding of the five proteins mix. Second, we deciphered the dynamics of each protein partner in the apo and holo states from HDX labelling experiments and performed data interpretation integrating dynamics information from HDX-MS and high-resolution structure from cryo-EM. This work highlights the complementary of both techniques for deep GPCR-Protein G coupling understanding in a drug discovery context.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jérôme CASTEL  

LAB HEAD: Thomas BOTZANOWSKI, HDX-MS lead

PROVIDER: PXD047827 | Pride | 2025-09-08

REPOSITORIES: Pride