Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Highly active and selective endopeptidases with programmed substrate specificities

Nature Chemical Biology volume 4, pages 290–294 (2008)Cite this article

Abstract

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (kcat/KM > 104 M−1 s−1). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu↓Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Screening methodology and the substrates used for screening.
Figure 2: Flow cytometric data.
Figure 3: Summary of sequence randomization strategy and library screening leading to the isolation of highly active and selective OmpT variants.

Similar content being viewed by others

References

  1. Overall, C.M. & Blobel, C.P. In search of partners: linking extracellular proteases to substrates. Nat. Rev. Mol. Cell Biol. 8, 245–257 (2007).

    Article  CAS  Google Scholar 

  2. Kuranaga, E. & Miura, M. Nonapoptotic functions of caspases: caspases as regulatory molecules for immunity and cell-fate determination. Trends Cell Biol. 17, 135–144 (2007).

    Article  CAS  Google Scholar 

  3. Page-McCaw, A., Ewald, A.J. & Werb, Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat. Rev. Mol. Cell Biol. 8, 221–233 (2007).

    Article  CAS  Google Scholar 

  4. Yamada, T., Shimada, Y. & Kikuchi, M. Integrin-specific tissue-type plasminogen activator engineered by introduction of the Arg-Gly-Asp sequence. Biochem. Biophys. Res. Commun. 228, 306–311 (1996).

    Article  CAS  Google Scholar 

  5. Jenny, R.J., Mann, K.G. & Lundblad, R.L. A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein Expr. Purif. 31, 1–11 (2003).

    Article  CAS  Google Scholar 

  6. Turkina, M.V. & Vener, A.V. Identification of phosphorylated proteins. Methods Mol. Biol. 355, 305–316 (2007).

    CAS  PubMed  Google Scholar 

  7. Marnett, A.B. & Craik, C.S. Papa's got a brand new tag: advances in identification of proteases and their substrates. Trends Biotechnol. 23, 59–64 (2005).

    Article  CAS  Google Scholar 

  8. Ballinger, M.D., Tom, J. & Wells, J.A. Furilisin: a variant of subtilisin BPN' engineered for cleaving tribasic substrates. Biochemistry 35, 13579–13585 (1996).

    Article  CAS  Google Scholar 

  9. Hedstrom, L., Szilagyi, L. & Rutter, W.J. Converting trypsin to chymotrypsin: the role of surface loops. Science 255, 1249–1253 (1992).

    Article  CAS  Google Scholar 

  10. Hopfner, K.P. et al. New enzyme lineages by subdomain shuffling. Proc. Natl. Acad. Sci. USA 95, 9813–9818 (1998).

    Article  CAS  Google Scholar 

  11. Khersonsky, O., Roodveldt, C. & Tawfik, D.S. Enzyme promiscuity: evolutionary and mechanistic aspects. Curr. Opin. Chem. Biol. 10, 498–508 (2006).

    Article  CAS  Google Scholar 

  12. Kukkonen, M. & Korhonen, T.K. The omptin family of enterobacterial surface proteases/adhesins: from housekeeping in Escherichia coli to systemic spread of Yersinia pestis. Int. J. Med. Microbiol. 294, 7–14 (2004).

    Article  CAS  Google Scholar 

  13. McCarter, J.D. et al. Substrate specificity of the Escherichia coli outer membrane protease OmpT. J. Bacteriol. 186, 5919–5925 (2004).

    Article  CAS  Google Scholar 

  14. Walton, T.A. & Sousa, M.C. Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation. Mol. Cell 15, 367–374 (2004).

    Article  CAS  Google Scholar 

  15. Varadarajan, N., Gam, J., Olsen, M.J., Georgiou, G. & Iverson, B.L. Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity. Proc. Natl. Acad. Sci. USA 102, 6855–6860 (2005).

    Article  CAS  Google Scholar 

  16. Hwang, J.K. & Warshel, A. Why ion pair reversal by protein engineering is unlikely to succeed. Nature 334, 270–272 (1988).

    Article  CAS  Google Scholar 

  17. Graf, L. et al. Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin. Biochemistry 26, 2616–2623 (1987).

    Article  CAS  Google Scholar 

  18. Caputo, A., Parrish, J.C., James, M.N., Powers, J.C. & Bleackley, R.C. Electrostatic reversal of serine proteinase substrate specificity. Proteins 35, 415–424 (1999).

    Article  CAS  Google Scholar 

  19. Vandeputte-Rutten, L. et al. Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site. EMBO J. 20, 5033–5039 (2001).

    Article  CAS  Google Scholar 

  20. Barrett, A.J.R., Neil, D. & Woessner, J.F. Handbook of Proteolytic Enzymes 1–1666 (Academic Press, London, 1998).

    Google Scholar 

  21. Hwang, B.Y. et al. Substrate specificity of the Escherichia coli outer membrane protease OmpP. J. Bacteriol. 189, 522–530 (2007).

    Article  CAS  Google Scholar 

  22. Dawson, K.M. et al. Plasminogen mutants activated by thrombin. Potential thrombus-selective thrombolytic agents. J. Biol. Chem. 269, 15989–15992 (1994).

    CAS  PubMed  Google Scholar 

  23. Gardell, S.J. et al. Isolation, characterization, and cDNA cloning of a vampire bat salivary plasminogen activator. J. Biol. Chem. 264, 17947–17952 (1989).

    CAS  PubMed  Google Scholar 

  24. Peschel, A. & Sahl, H.G. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nat. Rev. Microbiol. 4, 529–536 (2006).

    Article  CAS  Google Scholar 

  25. Stumpe, S., Schmid, R., Stephens, D.L., Georgiou, G. & Bakker, E.P. Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of Escherichia coli. J. Bacteriol. 180, 4002–4006 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Park, H.S. et al. Design and evolution of new catalytic activity with an existing protein scaffold. Science 311, 535–538 (2006).

    Article  CAS  Google Scholar 

  27. Collins, C.H., Leadbetter, J.R. & Arnold, F.H. Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR. Nat. Biotechnol. 24, 708–712 (2006).

    Article  CAS  Google Scholar 

  28. Santoro, S.W., Wang, L., Herberich, B., King, D.S. & Schultz, P.G. An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nat. Biotechnol. 20, 1044–1048 (2002).

    Article  CAS  Google Scholar 

  29. Segers, K., Dahlback, B. & Nicolaes, G.A. Coagulation factor V and thrombophilia: background and mechanisms. Thromb. Haemost. 98, 530–542 (2007).

    Article  CAS  Google Scholar 

  30. Grodberg, J., Lundrigan, M.D., Toledo, D.L., Mangel, W.F. & Dunn, J.J. Complete nucleotide sequence and deduced amino acid sequence of the ompT gene of Escherichia coli K-12. Nucleic Acids Res. 16, 1209 (1988).

    Article  CAS  Google Scholar 

  31. Fromant, M., Blanquet, S. & Plateau, P. Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Anal. Biochem. 224, 347–353 (1995).

    Article  CAS  Google Scholar 

  32. Hoover, D.M., Wu, Z., Tucker, K., Lu, W. & Lubkowski, J. Antimicrobial characterization of human beta-defensin 3 derivatives. Antimicrob. Agents Chemother. 47, 2804–2809 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Skretas and J. Link for reading the manuscript, J. Borrock and M. Pogson for assistance in preparing the graphical abstract and K. Griswold for many helpful discussions. We also thank E. Farinas for the initial design of the primers. This work was supported by US National Institutes of Health grants R01 GM065551 and RO1 GM073089.

Author information

Authors and Affiliations

  1. Institute for Cell and Molecular Biology, University of Texas, Austin, 78712, Texas, USA

    Navin Varadarajan, George Georgiou & Brent L Iverson

  2. Department of Chemical Engineering, University of Texas, Austin, 78712, Texas, USA

    Navin Varadarajan, Bum-Yeol Hwang & George Georgiou

  3. Department of Chemistry and Biochemistry, University of Texas, Austin, 78712, Texas, USA

    Sarah Rodriguez & Brent L Iverson

Authors
  1. Navin Varadarajan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bum-Yeol Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. George Georgiou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brent L Iverson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.V., B.L.I. and G.G. designed the research. B.-Y.H. ran the substrate-phage experiments; N.V. ran all the other experiments. S.R. assisted N.V. with library construction and kinetic characterization. N.V., B.L.I. and G.G. wrote the manuscript.

Corresponding authors

Correspondence to George Georgiou or Brent L Iverson.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 354 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Varadarajan, N., Rodriguez, S., Hwang, BY. et al. Highly active and selective endopeptidases with programmed substrate specificities. Nat Chem Biol 4, 290–294 (2008). https://doi.org/10.1038/nchembio.80

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.80

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing