Structure-Function Analysis of Yeast Grx5 Monothiol Glutaredoxin Defines Essential Amino Acids for the Function of the Protein

Abstract

Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/ sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys 60 and Gly 61 are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly 115 and Gly 116 could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys 117 . Substitution of Phe 50 alters the ␤-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.Glutaredoxins are thiol oxidireductases that catalyze redox reactions involving reduced glutathione as a hydrogen donor for the reduction of protein disulfides (dithiol mechanism of action) or glutathione-protein-mixed disulfides (monothiol mechanism of action) (see Refs. 1 and 2 for review). Previously described glutaredoxins are small proteins (about 10 kDa) with a conserved active site that includes two cysteine residues (Cys-Pro-Tyr-Cys). Site-directed mutagenesis (3-5) has demonstrated that both cysteine residues in the active site are required for the dithiol reaction. In contrast, the amino-terminal cysteine is sufficient to catalyze the deglutathionylation of the reduced glutathione-mixed disulfides that are formed under oxidative stress conditions (5).Three-dimensional structures of oxidized and reduced forms of viral, bacterial, and mammalian glutaredoxins and also of reduced glutathione-glutaredoxin complexes have been identified using x-ray crystallography (6, 7) or nuclear magnetic resonance spectroscopy (8 -14). These studies have revealed which residues, apart from those at the active site, are important for stable interactions between glutathione and the glutaredoxin molecule (10,13,14). Dithiol glutaredoxins are members of the thioredoxin superfamily (15, 16) along with at least five other classes of proteins that interact with cysteine-containing substrates (thioredoxins, DbsA, protein disulfide isomerases, glutathione S-transferases, and glutathione peroxidases). This superfamily shares a structural motif (called the thioredoxin fold or ␣␤␣ fold) formed by a four or fivestranded ␤-sheet (with parallel and antiparallel strands) surrounded by three or more ␣-helices distributed on ei...

AtGRXcp, an Arabidopsis Chloroplastic Glutaredoxin, Is Critical for Protection against Protein Oxidative Damage

“…6). In this case, our findings are similar to that observed with the Phe 62 variants in yeast Grx5 (47). Recently, the resolved three-dimensional solution structure of the bacterial Grx4 reveals that this monothiol Grx has a unique structure, which is significantly different from the dithiol Grxs (53).…”

“…5, B-D). In contrast, mutation of both the conserved Cys 60 and the nonconserved Cys 117 in yeast Grx5 and the conserved Cys 30 in bacterial Grx4 did not significantly affect protein stability (20,47,52). These findings suggest that this particular cysteine residue (Cys 97 ) may play a unique role in the plant AtGRXcp.…”

Structure-Function Relationship of the Chloroplastic Glutaredoxin S12 with an Atypical WCSYS Active Site

“…Some CGFS Grxs have been reported to possess a disulfide bridge involving an extra active site cysteine at the same position as in GrxS12 (5-7). For example, in S. cerevisiae Grx5, the importance of this disulfide is not clear, since it is required in vitro for deglutathionylation activity but not in vivo for the iron-sulfur biogenesis (48). For C. reinhardtii Grx3, it is also crucial in vitro, acting as a resolving cysteine for deglutathionylation reactions and then for its ferredoxin thioredoxin reductase-dependent regeneration (7).…”

Comparative genomics of yeast species: new insights into their biology

“…Function acquisition can also result from new combinations of protein domains, for example, as revealed in the S. cerevisiae genome by monothiol glutaredoxins, studied by our group [3]. This is a family of three proteins, one of which is mitochondrial (Grx5) and is involved in the synthesis of iron-sulfur clusters, while the other two (Grx3 and Grx4) are nuclear and their functions are unknown.…”

“…2) by using paired sequence reads from both ends Experimental and unfinished genomic sequence Fig. 1 The hypothetical evolution of monothiol glutaredoxins based on results described in [3] and on sequence comparisons (see text for more details). Grx Monothiol glutaredoxin domain, Trx thioredoxin domain, NLS nuclear localization sequence.…”