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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Lewinsohn, R. (1981). Carlos Chagas and the discovery of Chagas’ disease (American trypanosomiasis). Journal of the Royal Society of Medicine, 74(6), 451–455.
W. H. O. (2015). Chagas disease in Latin America: An epidemiological update based on 2010 estimates. Weekly Epidemiological Record, 90(6), 33–44.
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.
Bonney, K. M., & Engman, D. M. (2008). Chagas heart disease pathogenesis: One mechanism or many? Current Molecular Medicine, 8(6), 510–518.
Rassi, A., & Marin-Neto, J. A. (2010). Chagas disease. Lancet, 375(9723), 1388–1402.
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.
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.
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.
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.
Sajid, M., & McKerrow, J. H. (2002). Cysteine proteases of parasitic organisms. Molecular and Biochemical Parasitology, 120(1), 1–21.
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.
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.
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.
Ishidoh, K., & Kominami, E. (2002). Processing and activation of lysosomal proteinases. Biological Chemistry, 383(12), 1827–1831.
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.
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.
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.
Tina, K. G., B., R., & S., N. (2007). Protein Interactions Calculator. Nucleic Acids Research, 35, W473–W476.
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.
PyMOL Molecular Graphics System. Version 1.8. Schrödinger, LLC.
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.
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.
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.
Eder, J., Rheinnecker, M., & Fersht, R. (1993). Folding of subtilisin BPN’: Role of the pro-sequence. Journal of Molecular Biology, 32(1), 18–26.
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.
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.
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.
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.
Conflict of interest
The authors declare that they have no competing interests.
Marisol Serrano and Veronica Gonzalez contributed equally to this work.
Electronic supplementary material
About this article
Cite this article
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). https://doi.org/10.1007/s12013-017-0816-3
- Cysteine protease
- Chagas disease
- Trypanosoma cruzi
- Circular dichroism