Despite advances
in characterizing the structures and
functions
of G protein-coupled receptors (GPCRs), our understanding of GPCR
activation and signaling is still limited by the lack of information
on conformational dynamics. It is particularly challenging to study
the dynamics of GPCR complexes with their signaling partners because
of their transient nature and low stability. Here, by combining cross-linking
mass spectrometry (CLMS) with integrative structure modeling, we map
the conformational ensemble of an activated GPCR-G protein complex
at near-atomic resolution. The integrative structures describe heterogeneous
conformations for a high number of potential alternative active states
of the GLP-1 receptor–Gscomplex. These structures
show marked differences from the previously determined cryo-EM structure,
especially at the receptor–Gsinterface and in the
interior of the Gsheterotrimer. Alanine-scanning mutagenesis
coupled with pharmacological assays validates the functional significance
of 24 interface residue contacts only observed in the integrative
structures, yet absent in the cryo-EM structure. Through the integration
of spatial connectivity data from CLMS with structure modeling, our
study provides a new approach that is generalizable to characterizing
the conformational dynamics of GPCR signaling complexes.