Recent studies showed that several widely used glucagon-like peptide-1 receptor agonists (GLP-1RAs) can self-assemble into dynamic oligomeric species and ultimately less-regular, non-amyloid aggregates. While mature aggregates can be characterized by high-resolution microscopy methods, probing the conformational dynamics of early-stage oligomeric species remains challenging, which limits our understanding of how the misfolding and aggregation propensity of GLP-1RAs is modulated by physiologically relevant conditions such as protein concentration or the presence of metal ions. In this work, multiple structural mass spectrometry (MS) methods were used in combination to resolve the earliest molecular changes that lead to semaglutide oligomerization. These structural MS methods together reveal important information such as the heterogeneity and stoichiometry of oligomers, their structural/thermal stability, protein conformational changes, the molecular interface for self-assembly, relative monomer orientation in oligomers, and aggregation pathway of the resulting oligomeric species, both with and without metal ions present. We found that semaglutide forms oligomers up to 10-mers within 24 h; among seven essential metals for humans, low micromolar concentrations of Cu2+ and Zn2+ ions drive the formation of higher-order and more abundant oligomeric species, an effect attributed to metal binding-mediated stabilization of small oligomers. Oligomeric species are associated via hydrophobic C-termini in a parallel orientation. N-termini and the lipid chain are, instead, loosely engaged and available for high-affinity binding to human serum albumin (HSA), an interaction that facilitates the dissociation of oligomeric species and is further enhanced when HSA is glycated. Our findings underscore the power of structural MS to track early-stage oligomerization.
