Deciphering molecular determinants of GPCR-G protein receptor interactions by complementary integrative structural biology methods

Castel, J.; Botzanowski, T.; Brooks, I.; Frechard, A.; Bey, G.; Schroeter, M.; Del Nero, E.; Debaene, F.; Ciesielski, F.; Zeyer, D.; Cianferani, S.; Morales, R.

doi:10.1101/2024.10.21.619395

bioRxiv · 10.1101/2024.10.21.619395

Deciphering molecular determinants of GPCR-G protein receptor interactions by complementary integrative structural biology methods

Castel, J.·Botzanowski, T.·Brooks, I.·Frechard, A.·Bey, G.·Schroeter, M.·Del Nero, E.·Debaene, F.·Ciesielski, F.·Zeyer, D.·Cianferani, S.·Morales, R.

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Abstract

Many physiological processes are dependent on G protein-coupled receptors (GPCRs), the biggest family of human membrane proteins and a significant class of therapeutic targets. Once activated by external stimuli, GPCRs use G proteins and arrestins as transducers to generate second messengers and trigger downstream signaling, leading to diverse signaling profiles. The G protein-coupled bile acid receptor 1 (GPBAR1, also known as Takeda G protein-coupled receptor 5, TGR5) is a class A bile acid membrane receptor that regulates energy homeostasis and glucose and lipid metabolism. GPBAR1/G protein interactions are implicated in the prevention of diabetes and the reduction of inflammatory responses, making GPBAR1 a potential therapeutic target for metabolic disorders. Here, we present an integrated structural biology approach combining hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) to identify the molecular determinants of GPBAR1 conformational dynamics upon G protein binding. Thanks to extensive optimization of both HDX-MS and cryo-EM workflows, we achieved over 99% sequence coverage along with a 2.5-[A] resolution structure, both of which are state-of-the-art and solely obtained for complete GPCR complexes. Altogether, our results provide information on the under-investigated GPBAR1 binding mode to its cognate G protein, pinpointing the synergic and powerful combination of higher (cryo-EM) and lower (HDX-MS) resolution structural biology techniques to dissect GPCR/G protein binding characteristics. Short statementThis work highlights the utility of integrating cryo-EM and HDX-MS for studying large multiprotein complexes such as GPCR/G protein complexes. Cryo-EM offers high-resolution structural details, while HDX-MS reveals the dynamic conformational changes during assembly, providing a comprehensive structural view of difficult-to-study membrane protein systems.

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Castel, J., Botzanowski, T., Brooks, I., Frechard, A., Bey, G., Schroeter, M., Del Nero, E., Debaene, F., Ciesielski, F., Zeyer, D., Cianferani, S., Morales, R.. 2024-10-24. Deciphering molecular determinants of GPCR-G protein receptor interactions by complementary integrative structural biology methods. https://doi.org/10.1101/2024.10.21.619395

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