The high resolution crystal structure of recombinant Crithidia fasciculatatryparedoxin-I

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase invo lved in the regulation of oxidative stress in parasitic trypanosomatids, Th e crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dis persion methods applied to a selenomethionyl derivative. The model comprise s residues 3 to 145 with 236 water molecules and has been refined using all data between a 19- and 1.4-Angstrom resolution to an R-factor and R-free o f 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence ide ntity, tryparedoxin-I presents a five-stranded twisted beta-sheet and two e lements of helical structure in the same type of fold as displayed by thior edoxin, the archetypal thiol disulfide oxidoreductase, However, the relatio nship of secondary structure with the linear amino acid sequences is differ ent for each protein, producing a distinctive topology. The beta-sheet core is extended in the trypanosomatid protein with an N-terminal beta-hairpin, There are also differences in the content and orientation of helical eleme nts of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredox in and tryparedoxin-I. A right-handed redox-active disulfide is formed betw een Cys-40 and Cys-43 at the N-terminal region of a distorted cu-helix (arl ), Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity, Three C-H ... O hydrogen bonds donated from two proline residues se rve to stabilize the disulfide-carrying helix and support the correct align ment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and prov ides a template for the discovery or design of selective inhibitors of hydr operoxide metabolism in trypanosomes. Such inhibitors are sought as potenti al therapies against a range of human pathogens.