Identification of Structure-Stabilizing Interactions in Enzymes: A Novel Mechanism to Impact Enzyme Activity


Cruzain, a cysteine protease in the cathepsin family, is pivotal to the life-cycle of Trypanosoma cruzi, the etiological agent in Chagas disease. Current inhibitors of cruzain suffer from drawbacks involving gastrointestinal and neurological side effects and as a result have spurred the search for alternative anti-trypanocidals. Through sequence alignment studies and intra-residue interaction analysis of the pro-protein of cruzain (pro-cruzain), we have identified a host of non-active site residues that are conserved among the cathepsins. We hypothesize that these conserved amino acids play a critical role in structure-stabilizing interactions among the cathepsins and are therefore crucial for eventually gaining protease activity. As predicted, mutation of selected conserved non-active site amino-acid candidates in cruzain resulted in a compromised structural stability and a corresponding loss in enzymatic activity relative to wild-type enzyme. By advancing the discovery of novel, non-active-site-based targets to arrest enzymatic activity our results potentially open the field of alternative inhibitor design. The advantages of defining such a non-active-site inhibitor design space is discussed.

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  1. 1.

    Lewinsohn, R. (1981). Carlos Chagas and the discovery of Chagas’ disease (American trypanosomiasis). Journal of the Royal Society of Medicine, 74(6), 451–455.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    W. H. O. (2015). Chagas disease in Latin America: An epidemiological update based on 2010 estimates. Weekly Epidemiological Record90(6), 33–44.

  3. 3.

    Who, F. (2010). Working to overcome the global impact of neglected tropical diseases First WHO report on neglected tropical diseases. World Health, 86(13), 1–184.

    Google Scholar 

  4. 4.

    Bonney, K. M., & Engman, D. M. (2008). Chagas heart disease pathogenesis: One mechanism or many? Current Molecular Medicine, 8(6), 510–518.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Rassi, A., & Marin-Neto, J. A. (2010). Chagas disease. Lancet, 375(9723), 1388–1402.

    Article  PubMed  Google Scholar 

  6. 6.

    Tanowitz, H. B., Kirchhoff, L. V., Simon, D., Morris, S. A., Weiss, L. M., & Wittner, M. (1968). Chagas’ disease. Proceedings of the Royal Society of Medicine, 5(5), 444–445.

    Google Scholar 

  7. 7.

    Cazzulo, J. J., Stoka, V., & Turk, V. (2001). The major cysteine proteinase of Trypanosoma cruzi: A valid target for chemotherapy of Chagas’ disease. Current Pharmaceutical Design, 7, 1143–1156.

    CAS  Article  Google Scholar 

  8. 8.

    McKerrow, J. H., Engel, J. C., & Caffrey, C. R. (1999). Cysteine protease inhibitors as chemotherapy for parasitic infections. Bioorganic & Medicinal Chemistry, 7(4), 639–644.

    CAS  Article  Google Scholar 

  9. 9.

    McKerrow, J. H., McGrath, M. E., & Engel, J. C. (1995). The cysteine protease of Trypanosoma cruzi as a model for antiparasite drug design. Parasitology Today, 11(8), 279–282.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Sajid, M., & McKerrow, J. H. (2002). Cysteine proteases of parasitic organisms. Molecular and Biochemical Parasitology, 120(1), 1–21.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Lima, L., Ortiz, P. A., Silva, F. M., Joao Marcelo, P., Alves, M. G. S., Alane, P., Cortez, S. C. A., Buck, G. A., & Teixeira, M. M. G. (2012). Repertoire, genealogy and genomic organization of cruzipain and homologous genes in trypanosoma cruzi, T. Cruzi-Like and Other Trypanosome Species. PLoS One, 7(6), 1–15.

    Google Scholar 

  12. 12.

    Rangel, H. A., Araújo, P. M., Repka, D., & Costa, M. G. (1981). Trypanosoma cruzi: isolation and characterization of a proteinase. Experimental Parasitology, 52(2), 199–209.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Eakin, A. E., Mills, A., Harth, G., Mckerrowo, J. H., & Craiks, C. S. (1992). The sequence, organization, and expression of the major cysteine protease (cruzain) from trypanosoma cruzi. The Journal of Biological Chemistry, 267(1990), 7411–7420.

    CAS  PubMed  Google Scholar 

  14. 14.

    Ishidoh, K., & Kominami, E. (2002). Processing and activation of lysosomal proteinases. Biological Chemistry, 383(12), 1827–1831.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., & Notredame, C. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7(1), 539.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5(4), 725–738.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Tina, K. G., B., R., & S., N. (2007). Protein Interactions Calculator. Nucleic Acids Research, 35, W473–W476.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Doyle, P. S., Zhou, Y. M., Engel, J. C., & McKerrow, J. H. (2007). A cysteine protease inhibitor cures Chagas’ disease in an immunodeficient-mouse model of infection. Antimicrobial Agents and Chemotherapy, 51(11), 3932–3939.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    PyMOL Molecular Graphics System. Version 1.8. Schrödinger, LLC.

  21. 21.

    Sivashanmugam, A., Murray, V., Cui, C., Zhang, Y., Wang, J., & Li, Q. (2009). Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Science, 18(1), 936–948.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Coulombe, R., Grochulski, P., Sivaraman, J., Ménard, R., Mort, J. S., & Cygler, M. (1996). Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment. The EMBO Journal, 15(20), 5492–5503.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Reis, F. C. G., Costa, T. F. R., Sulea, T., Mezzetti, A., Scharfstein, J., Brömme, D., & Lima, A. P. C. (2007). The propeptide of cruzipain-a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain, and of the human enzyme cathepsin F. The FEBS Journal, 274(5), 1224–1234.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Eder, J., Rheinnecker, M., & Fersht, R. (1993). Folding of subtilisin BPN’: Role of the pro-sequence. Journal of Molecular Biology, 32(1), 18–26.

    CAS  Google Scholar 

  25. 25.

    Lerner, C. G., Kobayashi, T., & Inouye, M. (1990). Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis. The Journal of Biological Chemistry, 265(33), 20085–20086.

    CAS  PubMed  Google Scholar 

  26. 26.

    Smith, S. M., & Gottesman, M. M. (1989). Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli. The Journal of Biological Chemistry, 264(34), 20487–20495.

    CAS  PubMed  Google Scholar 

  27. 27.

    Ruan, B., Hoskins, J., & Bryan, P. N. (1999). Rapid folding of calcium-free subtilisin by a stabilized pro-domain mutant. Biochemistry, 38(26), 8562–8571.

    CAS  Article  PubMed  Google Scholar 

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MN would like to thank the American Heart Association (National Scientist Development Grant) for the financial support. The authors acknowledge the Border Biomedical Research Center (BBRC) and the staff of the DNA Core Facility at the University of Texas at El Paso for services and facilities provided and the RISE Program. Some of this work was made possible due to support from NIGMS/NIH RL5GM118969, TL4GM118971, UL1GM118970. Denise Chavez and Research reported in this publication was supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM060424. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Correspondence to Mahesh Narayan.

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Marisol Serrano and Veronica Gonzalez contributed equally to this work.

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Serrano, M., Gonzalez, V., Ray, S. et al. Identification of Structure-Stabilizing Interactions in Enzymes: A Novel Mechanism to Impact Enzyme Activity. Cell Biochem Biophys 76, 59–71 (2018).

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  • Pro-cruzain
  • Cysteine protease
  • Expression
  • Chagas disease
  • Trypanosoma cruzi
  • Circular dichroism
  • Auto-activation