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Structural and kinetic determinants of protease substrates

Abstract

Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in α-helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.

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Figure 1: Experimental approach.
Figure 2: N-terminomics reveals protease-specific cleavage sites.
Figure 3: Distribution of amino acids in the P1 position of unascribed cleavage sites.
Figure 4: Specificity of caspase-3 and GluC.
Figure 5: Secondary-structure preferences of human caspase-3 and GluC.
Figure 6: Biochemical and kinetic analysis of cleavage sites identified by N-terminomics.
Figure 7: Engineered carA mutants are cleaved more efficiently than wild-type carA.
Figure 8: Natural human caspase-3 substrates are kinetically superior to E. coli substrates.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) Roadmap Initiative National Biotechnology Resource Center grant RR20843 for the Center on Proteolytic Pathways, CA69381 from the National Cancer Institute (NCI) and by Training Grant 5T32CA77109-9 from the NCI. We thank M. Enoksson for helpful discussions.

Author information

Authors and Affiliations

Authors

Contributions

J.C.T. designed and performed most experiments and interpreted results; W.Z. performed LC-MS/MS analysis and database searching; C.P. designed and performed Km determination experiments; T.R. prepared some N-terminomic samples; S.J.S. performed Edman degradation; A.M.E. provided advice and helped revise the manuscript; S.J.R. aided in structural interpretation; G.S.S. designed the scope of the study, interpreted results and, together with J.C.T. and S.J.R., wrote the manuscript.

Corresponding author

Correspondence to Guy S Salvesen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Table 5 (PDF 3156 kb)

Supplementary Table 1

N-terminome of caspase-3 treated E. coli lysate & controls (XLS 395 kb)

Supplementary Table 2

N-terminome of GluC treated E. coli lysate & controls (XLS 354 kb)

Supplementary Table 3

Caspase-3 cleavage-sites in the E. coli proteome determined by N-terminomics. (XLS 21 kb)

Supplementary Table 4

GluC cleavage-sites in the E. coli proteome determined by N-terminomics. (XLS 26 kb)

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Timmer, J., Zhu, W., Pop, C. et al. Structural and kinetic determinants of protease substrates. Nat Struct Mol Biol 16, 1101–1108 (2009). https://doi.org/10.1038/nsmb.1668

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