Structural Mass Spectrometry Captures Residue-Resolved Comprehensive Conformational Rearrangements of a G Protein-Coupled Receptor

• Published in: Journal of the American Chemical Society Vol. 146; no. 29; pp. 20045 - 20058
• Main Authors: Liu, Hongyue, Yan, Pengfei, Zhang, Zhaoyu, Han, Hongbo, Zhou, Qingtong, Zheng, Jie, Zhang, Jian, Xu, Fei, Shui, Wenqing
• Format: Journal Article
• Language: English
• Published: American Chemical Society 24.07.2024
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/jacs.4c03922

G protein–coupled receptor (GPCR) structural studies with in-solution spectroscopic approaches have offered distinctive insights into GPCR activation and signaling that highly complement those yielded from structural snapshots by crystallography or cryo-EM. While most current spectroscopic approaches allow for probing structural changes at selected residues or loop regions, they are not suitable for capturing a holistic view of GPCR conformational rearrangements across multiple domains. Herein, we develop an approach based on limited proteolysis mass spectrometry (LiP-MS) to simultaneously monitor conformational alterations of a large number of residues spanning both flexible loops and structured transmembrane domains for a given GPCR. To benchmark LiP-MS for GPCR conformational profiling, we studied the adenosine 2A receptor (A2AR) in response to different ligand binding (agonist/antagonist/allosteric modulators) and G protein coupling. Systematic and residue-resolved profiling of A2AR conformational rearrangements by LiP-MS precisely captures structural mechanisms in multiple domains underlying ligand engagement, receptor activation, and allostery, and may also reflect local conformational flexibility. Furthermore, these residue-resolution structural fingerprints of the A2AR protein allow us to readily classify ligands of different pharmacology and distinguish the G protein-coupled state. Thus, our study provides a new structural MS approach that would be generalizable to characterizing conformational transition and plasticity for challenging integral membrane proteins.