Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism.
Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.
Menchise
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Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, Groupe Biocristallographie, ESA 7036, Université Henri Poincaré-Nancy I, BP 239, 54506 Vandoeuvre-lès-Nancy Cedex, France.
Corbier
C
C
Didierjean
C
C
Saviano
M
M
Benedetti
E
E
Jacquot
J P
JP
Aubry
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D003198:Computer Simulation; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011522:Protons; D017386:Sequence Homology, Amino Acid; D054480:Thioredoxin h; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D011522:Protons; D054480:Thioredoxin h; D001224:Aspartic Acid; D013879:Thioredoxins