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.
KeywordsPro-cruzain Cysteine protease Expression Chagas disease Trypanosoma cruzi Circular dichroism Auto-activation
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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Conflict of interest
The authors declare that they have no competing interests.
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