Structural dynamics underlying agonist activation of a GLP-1R-Gs precoupled complex
G-protein-coupled receptors (GPCRs) act as allosteric transmembrane signalling machines, generating distinct cellular responses depending on the conformational states induced by ligand binding. The glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR central to insulin secretion and body-weight regulation, is a key therapeutic target for obesity-associated metabolic disease. Here, we used hydrogen-deuterium exchange mass spectrometry to characterize ligand-evoked structural dynamics within a pre-coupled GLP-1R-Gs protein complex. Non-peptide agonists Chu-128 and danuglipron elicited overlapping dynamic perturbation profiles, with distinct drug-specific effects within the transmembrane bundle. In contrast, the natural GLP-1 hormone produced a weaker stabilizing effect on receptor backbone dynamics, while its inactive metabolite exerted opposing localised destabilization. Notably, both peptides uniquely modulated the highly flexible G-protein switch III loop, a key mediator of downstream signalling. These findings pinpoint areas where structural dynamics shape agonist efficacy and facilitate functional dynamics-integrated drug discovery of non-peptide agonists. SignificanceThe development of non-peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) represents a major advance in metabolic therapeutics, addressing key limitations of current peptide-based incretin therapies while enabling improved control over receptor signalling and pharmacokinetic properties. The recent FDA approval of a first-in-class small-molecule oral GLP-1R agonist, LY3502970, highlights the translational potential of this approach. However, the molecular basis by which distinct ligands modulate GLP-1R conformational dynamics and signalling remains poorly understood. Here we report how non-peptide agonists (Chu-128 and danuglipron) and endogenous GLP-1 peptide and its inactive metabolite shape the structural dynamics of a pre-coupled GLP-1R-Gs complex, revealing patterns linked to receptor activation. Our findings provide insights that guide the rational design of next-generation GLP-1R therapeutics.