Aziridination-Based Crosslinking Enables Site-Resolved Elucidation of Lipid Binding and Protein Conformational Changes.

Lipid-protein interactions play critical roles in cellular function, yet their site-resolved characterization remains challenging due to the dynamic nature of lipid binding and the limited availability of chemoselective strategies for covalently capturing native lipid-protein contacts. Here, we introduce aziridination-based lipid-protein crosslinking coupled with mass spectrometry (Azi-LPXL-MS), a chemoselective strategy that installs an N-H aziridine moiety onto acyl chains of native unsaturated lipids and enables subsequent crosslinking to proximal lysine residues using the amine-reactive reagent disuccinimidyl suberate (DSS). This integrated workflow simultaneously identifies lipid-binding sites and intraprotein peptide-peptide distance constraints, providing direct insight into lipid binding and binding-induced conformational changes within a single experiment. Application of Azi-LPXL-MS to β-lactoglobulin (BLG) demonstrates these capabilities, consistent with known structural models, while additionally providing solution-phase and conformational information not accessible from static structures alone. The power of the method is further demonstrated in the integral membrane enzyme diacylglycerol acyltransferase 1 (DGAT1). Azi-LPXL-MS reveals phosphatidic acid (PA)-binding sites and maps lipid-dependent conformational rearrangements across both resolved and previously uncharacterized regions. These data further define an allosteric conformational network linking lipid engagement to distal structural changes. Integrated with site-directed mutagenesis and molecular docking, two potential PA-binding sites in DGAT1, located in a surface pocket and an internal chamber, were identified and functionally validated, further supporting the experimental findings. These results establish Azi-LPXL-MS as a site-resolved method for studying lipid-protein interactions, providing insight into how individual lipid species engage proteins and influence their conformations in biological systems.