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

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.