Aziridination-Based Crosslinking Enables Site-Resolved Elucidation of Lipid Binding and Protein Conformational Changes.
- 1Department of Chemistry, Texas A&M University, 580 Ross Street, College Station, Texas77843, United States.
- 2Department of Biochemistry and Biophysics, Texas A&M University, 300 Olsen Boulevard, College Station, Texas77843, United States.
Abstract
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.
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BibTeX
@article{tsai2026aziridinationbased,
title = {{Aziridination-Based Crosslinking Enables Site-Resolved Elucidation of Lipid Binding and Protein Conformational Changes.}},
author = {Chia-Lung Tsai and Atiya Tahira Tasnim and Zhenyu Xi and Syuan-Ting Kuo and Gopal Reddy Ramidi and Jiaxin Feng and Michael Ryan Eastabrook and David H Russell and Xuewu Sui and Xin Yan},
journal = {Journal of the American Chemical Society},
year = {2026},
month = oct,
doi = {10.1021/jacs.6c08027},
url = {https://europepmc.org/article/MED/42912306},
note = {CC BY},
}
RIS
TY - JOUR
TI - Aziridination-Based Crosslinking Enables Site-Resolved Elucidation of Lipid Binding and Protein Conformational Changes.
AU - Chia-Lung Tsai
AU - Atiya Tahira Tasnim
AU - Zhenyu Xi
AU - Syuan-Ting Kuo
AU - Gopal Reddy Ramidi
AU - Jiaxin Feng
AU - Michael Ryan Eastabrook
AU - David H Russell
AU - Xuewu Sui
AU - Xin Yan
PY - 2026
DA - 2026/10/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c08027
UR - https://europepmc.org/article/MED/42912306
AB - 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.
SN - 0002-7863
SN - 1520-5126
ER -