Structure of trypanothione reductase from Crithidia fasciculata at 2.6 Å resolution; enzyme–NADP interactions at 2.8 Å resolution

Abstract

Trypanothione reductase is an FAD-dependent disulfide oxidoreductase which catalyses the reduction of trypanothione using NADPH as co-factor. The enzyme is unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved antitrypanocidal drugs. We present details of the structure of trypanothione reductase from Crithidia fasciculata solved by molecular replacement, using human glutathione reductase as a search model, and refined to an R factor of 16.1% with data between 8.0 and 2.6 A resolution. The model comprises two subunits (one containing 487 residues, the other 486), an FAD prosthetic group, plus 392 solvent molecules. The last four C-terminal residues are not seen in either subunit and the density is poor for the N-terminal residue of subunit B. The model has a root-mean-square deviation from ideality of 0.016 A for bond lengths and 3.2 degrees for bond angles. Each subunit was independently refined in the latter stages of the analysis but the subunits remain similar as indicated by the root-mean-square deviation of 0.35 A for C(alpha) atoms. Trypanothione reductase has 36% sequence identity with human glutathione reductase and the root-mean-square deviation between the 462 C(alpha) atoms in the secondary structural units common to the two proteins is 1.1 A. However, there are large differences in the loop regions and significant shifts in the orientation of the four domains within each subunit. Domain II, which binds the dinucleotide co-factor, and domain IV, which forms the interface between the two subunits, are both rotated by approximately 5 degrees with respect to domain I, which binds the FAD moiety, when compared with glutathione reductase. Crystals of trypanothione reductase have been soaked in the dinucleotide co-factor NADPH and N(1)-glutathionylspermidine disulfide substrate and the structure of the resulting complex determined at 2.8 A resolution. Strong density is observed for the adenosine end of the co-factor which forms many charged interactions with the protein though the density for the nicotinamide moiety is more diffuse. The mode of binding indicates that NADP is bound to the enzyme in a similar conformation to that observed with human glutathione reductase.

Modeled structure of trypanothione reductase of Leishmania infantum

“…Eleven amino-acid residues of domain I of TR from Trypanosoma directly interact with the FAD molecule via 17 interactions (10), with Met333, Gly196, Arg222, Arg228, Tyr198, and Tyr 221 involved in NADPH binding (10). These residues are conserved in TR of L. infantum.…”

A comparison of two independently determined structures of trypanothione reductase from Crithidia fasciculata

“…'Cold' regions (where atoms have lower thermal parameters) are found within the bulk of the enzyme, especially at the bottom of the disulfide substrate-binding site cleft. As detailed by Bailey et al (1994) this indicates that this area is consistently rigid. 'Hot' sections, with the higher thermal parameters are located in surface loops, C-or N-terminal residues and at the rim of the active-site cleft.…”

“…Details of the crystal packing in TRT are presented by Bailey et al (1994). Table 1 presents a summary of the experimental procedures used in the structure determinations for each form.…”

“…The disulfide-binding site is seen as a cleft, of about 20 ik in width by 15 A deep, at the subunit interface, and is composed of residues from central and FAD-binding domains of one subunit and the interface domain of the other. A full description is given by Bailey, Fairlamb & Hunter (1994).…”

“…The TR from C. fasciculata has been studied independently in two crystal forms. A monoclinic form at Rockefeller University, NY (Kuriyan et al, 1990(Kuriyan et al, , 1991, Protein Data Bank (PDB) accession code 2TPR (Bernstein et al, 1977) and a tetragonal form at the University of Manchester, England (Bailey et al, 1994, PDB accession code 1TYT).…”