Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 44, Pages 11162-11167Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1813574115
Keywords
chemical cross-linker; sulfur-fluoride exchange; cross-linking mass spectrometry; proximity-enhanced reactivity; protein-protein interaction
Categories
Funding
- NIH [5TL1TR001871, GM122603, P01AG002132, R01GM118384, RF1MH114079]
- NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES [TL1TR001871] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM118384, R35GM122603] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF MENTAL HEALTH [RF1MH114079] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [P01AG002132] Funding Source: NIH RePORTER
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Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a plant-and-cast cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues planting the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then cast across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximityenhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical crosslinking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.
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