Abstract

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein–protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

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Acknowledgements

This work was supported by the National Institutes of Health (CA087660, CA132630 (to B.F.C.), GM108208 (to K.M.B.) and GM069832 (to S.F.)) and the Deutsche Forschungsgemeinschaft (to S.M.H.). The authors thank M. Dix and M. Radu Suciu for providing assistance with proteomics data collection and analysis, respectively. The authors acknowledge PhosphoSitePlus (www.phosphosite.org) and the Scripps NMR and MS core facilities.

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Author notes

    • Stephan M. Hacker
    •  & Keriann M. Backus

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92307, USA

    • Stephan M. Hacker
    • , Keriann M. Backus
    • , Michael R. Lazear
    •  & Benjamin F. Cravatt
  2. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92307, USA

    • Stefano Forli
  3. Laboratory of Protein Design & Immunoengineering, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland

    • Bruno E. Correia

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Contributions

S.M.H., K.M.B. and B.F.C. conceived of the project, designed experiments and analysed data. S.M.H. and K.M.B. performed mass spectrometry experiments and data analysis. S.M.H. synthesized compounds, cloned, expressed and purified proteins, and conducted inhibition studies. K.M.B., M.R.L. and B.E.C. wrote software. S.M.H. and K.M.B. compiled and analysed mass spectrometry data. S.F. wrote software and conducted computational analyses. M.R.L. conducted immunoprecipitation studies and data analysis. S.M.H., K.M.B. and B.F.C. wrote the manuscript.

Competing interests

The authors declare competing financial interests. B.F.C. is a founder and advisor to Vividion Therapeutics, a biotechnology company interested in using chemical proteomic methods to develop small-molecule drugs to treat human disease.

Corresponding authors

Correspondence to Stephan M. Hacker or Keriann M. Backus or Benjamin F. Cravatt.

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https://doi.org/10.1038/nchem.2826

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