The High Resolution Crystal Structure of Recombinant Crithidia fasciculata Tryparedoxin-I

Abstract

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19-and 1.4-Å resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted ␤-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The ␤-sheet core is extended in the trypanosomatid protein with an N-terminal ␤-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A righthanded redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted ␣-helix (␣1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H⅐⅐⅐O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.Parasitic trypanosomatids, belonging to the order Kinetoplastida, cause debilitating and life-threatening human diseases such as African sleeping sickness, Chagas' disease, and the leishmaniases (1). The current therapies against these infections are inadequate due to poor drug efficacy and toxicity combined with increasing drug resistance (2). There is therefore an urgent need to understand how drugs already in use function so that they might be improved and to identify new targets for chemotherapeutic attack. The ideal target is an enzyme of a metabolic pathway that is essential for the survival of the parasite and either absent in the human host or one that presents differing substrate specificities (3). Because trypanosomatids are susceptible to oxidative stress, this aspect of their metabolism represents an attractive target for the development of new trypanocidal agents (4 -6).As with other organisms living in an aerobic environment, trypanosomes are exposed to reactive oxygen intermediates such as superoxide anion and hydrogen peroxide. These potentially destructive c...

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