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Engineering peptide ligase specificity by proteomic identification of ligation sites

Nature Chemical Biology volume 14, pages 50–57 (2018)Cite this article

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Abstract

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

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Figure 1: PILS applied to comprehensive characterization of subtiligase prime-side specificity.
Figure 2: Defining and re-engineering subtiligase specificity for P1′ and P2′ residues.
Figure 3: Scope of subtiligase-catalyzed N-terminal modification of folded proteins.
Figure 4: Modular strategy for subtiligase-catalyzed protein bioconjugation.
Figure 5: Algorithmically selected subtiligase cocktails for cellular N terminomics.

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Acknowledgements

We thank S. Coyle, Z. Hill, M. Hornsby, H. Huang, O. Julien, P. Lee, D. Sashital, L. Pack, A. Stewart, H. Tran, K. Wypysniak and members of the Wells laboratory for helpful discussions. We thank P. Lee and M. Hornsby (University of California, San Francisco) for the αGFP rAb expression vector, S. Pollock (University of California, San Francisco) for the HEK-293T-GFP cell line, and H. Tran (University of California, San Francisco) for the subtiligase E. coli expression vector. This work was supported by NIH grant 5R01GM081051-09 and the Harry and Dianna Hind Professorship in Pharmaceutical Sciences (to J.A.W.). A.M.W. is a Merck Fellow of the Helen Hay Whitney Foundation (F-1112) and holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund (1017065).

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  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA

    Amy M Weeks & James A Wells

  2. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA

    James A Wells

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Correspondence to James A Wells.

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A.M.W., J.A.W. and the Regents of the University of California have filed a patent application (US Provisional Patent Application No. 62/398,898) related to engineered subtiligase variants.

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Weeks, A., Wells, J. Engineering peptide ligase specificity by proteomic identification of ligation sites. Nat Chem Biol 14, 50–57 (2018). https://doi.org/10.1038/nchembio.2521

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