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• W2062917114 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. Cys60 and Gly61 are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly115 and Gly116 could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys117. Substitution of Phe50 alters the β-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. 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. Cys60 and Gly61 are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly115 and Gly116 could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys117. Substitution of Phe50 alters the β-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. protein kinase C-interacting cousin of thioredoxin 1% yeast extract, 2% peptone, and 2% dextrose 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. 1Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar and 2Holmgren A. Aslund F. Methods Enzymol. 1995; 252: 283-292Crossref PubMed Scopus (298) Google Scholar 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 (3Nikkola M. Gleason F.L. Saarinen M. Joelson T. Björnberg O. Eklund H. J. Biol. Chem. 1991; 266: 16105-16112Abstract Full Text PDF PubMed Google Scholar, 4Yang Y. Wells W.W. J. Biol. Chem. 1991; 266: 12759-12765Abstract Full Text PDF PubMed Google Scholar, 5Bushweller J.H. Aslund F. Wuthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar) 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 (5Bushweller J.H. Aslund F. Wuthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar). 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 (6Eklund H. Ingelman M. Söderberg B.-O. Uhlin T. Nordlund P. Nikkola M. Sonnerstam U. Joelsson T. Petratos K. J. Mol. Biol. 1992; 228: 596-618Crossref PubMed Scopus (68) Google Scholar, 7Katti S.K. Robbins A.H. Yang Y. Wells W.W. Protein Sci. 1995; 4: 1998-2005Crossref PubMed Scopus (62) Google Scholar) or nuclear magnetic resonance spectroscopy (8Sodano P. Xia T. Bushweller J.H. Björnberg O. Holmgren A. Billeter M. Wüthrich K. J. Mol. Biol. 1991; 221: 1311-1324Crossref PubMed Scopus (91) Google Scholar, 9Xia T. Bushweller J.H. Sodano P. Billeter M. Björnberg O. Holmgren A. Wüthrich K. Protein Sci. 1992; 1: 310-321Crossref PubMed Scopus (114) Google Scholar, 10Bushweller J.H. Billeter M. Holmgren A. Wüthrich K. J. Mol. Biol. 1994; 235: 1585-1597Crossref PubMed Scopus (120) Google Scholar, 11Ingelman M. Nordlund P. Eklund H. FEBS Lett. 1995; 370: 209-211Crossref PubMed Scopus (15) Google Scholar, 12Aslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 13Sun C. Berardi M.J. Bushweller J.H. J. Mol. Biol. 1998; 280: 687-701Crossref PubMed Scopus (79) Google Scholar, 14Nordstrand K. Aslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar). These studies have revealed which residues, apart from those at the active site, are important for stable interactions between glutathione and the glutaredoxin molecule (10Bushweller J.H. Billeter M. Holmgren A. Wüthrich K. J. Mol. Biol. 1994; 235: 1585-1597Crossref PubMed Scopus (120) Google Scholar, 13Sun C. Berardi M.J. Bushweller J.H. J. Mol. Biol. 1998; 280: 687-701Crossref PubMed Scopus (79) Google Scholar, 14Nordstrand K. Aslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar). Dithiol glutaredoxins are members of the thioredoxin superfamily (15Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 16Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar) 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 αβα fold) formed by a four or five-stranded β-sheet (with parallel and antiparallel strands) surrounded by three or more α-helices distributed on either side of the β-sheet (15Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar,16Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). Thioredoxins share with glutaredoxins the ability to reduce disulfides, although the former directly use NADPH as hydrogen donor (1Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). Dithiol glutaredoxins participate in a large number of functions in prokaryotic and eukaryotic cells, including the activation of ribonucleotide reductase (17Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar) and 3′-phosphoadenylylsulfate reductase (18Lillig C.H. Prior A. Schwenn J.D. Aslund F. Ritz D. Vlamis-Gardikas A. Holmgren A. J. Biol. Chem. 1999; 274: 7695-7698Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), reduction of ascorbate (19Wells W.W., Xu, D.P. Yang Y.F. Rocque P.A. J. Biol. Chem. 1990; 265: 15361-15364Abstract Full Text PDF PubMed Google Scholar), regulation of the DNA binding activity of nuclear factors (20Bandyopadhyay S. Strake D.W. Mieyal J.J. Gronostajski R.M. J. Biol. Chem. 1998; 273: 392-397Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and neuronal protection against dopamine-induced apoptosis (21Daily D. Vlamis-Gardikas A. Offen D. Mittelman L. Melamed E. Holmgren A. Barzilai A. J. Biol. Chem. 2001; 276: 1335-1344Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22Daily D. Vlamis-Gardikas A. Offen D. Mittelman L. Melamed E. Holmgren A. Barzilai A. J. Biol. Chem. 2001; 276: 21618-21626Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). A family of threeSaccharomyces cerevisiae proteins (Grx3, Grx4, and Grx5) has been described (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar) that has significant homology with dithiol glutaredoxins, preferentially at the carboxyl-terminal region of the molecules. The absence of any of these proteins leads to a decrease in cellular glutaredoxin activity, even though they do not contain the conserved active site of classic dithiol glutaredoxins. Instead, these proteins contain the conserved Cys-Gly-Phe-Ser motif at the amino-terminal region (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). This is the only cysteine residue found in Grx3 and Grx4, whereas Grx5 has an additional cysteine at the carboxyl-terminal moiety. From these data, it has been proposed that Grx3, Grx4, and Grx5 constitute a family of monothiol glutaredoxins in yeast (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). However, although there is a high degree of homology among them, these three proteins seem to carry out different cellular functions: the absence of Grx5 causes dramatic sensitivity to oxidants and growth defects in minimal medium, whereas no clear phenotypes are observed when Grx3 or Grx4 is absent. More recently, it has been shown that Grx5 is located at the mitochondria and involved in the biogenesis of iron/sulfur clusters (24Rodrı́guez-Manzaneque M.T. Tamarit J. Bellı́ G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (394) Google Scholar). Accumulation of cellular iron when Grx5 is absent could lead to protein oxidation and sensitivity to external oxidants. Available data about Grx3 and Grx4 indicate that they are not located in the mitochondria (24Rodrı́guez-Manzaneque M.T. Tamarit J. Bellı́ G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (394) Google Scholar). Proteins homologous to yeast monothiol glutaredoxins exist in all types of organisms from bacteria to humans (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar, 25Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 26Rahlfs S. Fischer M. Becker K. J. Biol. Chem. 2001; 276: 37133-37140Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The human homologue (PICOT1 protein) has been proposed as a negative regulator of protein kinase C-θ in the pathway leading to activation of the activator protein 1 and nuclear factor κB transcription factors (27Witte S. Villalba M., Bi, K. Liu Y. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The conserved region has been termed PICOT homology domain, and in Grx5, it corresponds to the majority of the peptide (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar, 25Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Human PICOT, yeast Grx3 and Grx4, and other eukaryotic homologous proteins possess amino-terminal extensions of PICOT homology domain. These extensions have signatures characteristic of thioredoxins or dithiol glutaredoxins that do not encompass the oxidoreductase active site (25Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). All these observations support the differential roles displayed by monothiol glutaredoxins regardless of their structural similarities. In this work, we show that Grx5 defines a ubiquitous family of proteins whose members are present in most types of organisms and are characterized by the presence of a thioredoxin fold structure. We also demonstrate the essential biological roles of a number of conserved amino acid residues, such as a cysteine located at the previously proposed active site in the amino-terminal region and a pair of glycines in the carboxyl-terminal region. CML235 (MAT a ura3-52 leu2Δ1 his3Δ200) was used as wild-type strain. MML19 is an isogenicΔgrx5::kanMX4 derivative of CML235 (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). MML160 was obtained from the latter by chromosomal integration of the YIplac211 vector (integrative, LEU2 marker) (28Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 3065-3073Google Scholar). MML161 was constructed similarly, although a YIplac211-derived plasmid (pMM25) with GRX5 expressed under its own promoter was integrated at LEU2. Other strains listed in Table I resulted from integration of pMM25-derived plasmids carrying the indicated point mutations at the mutant leu2 locus of MML19.Table IYeast strains and inferred secondary structure at the amino acid substitution positionsStrainPlasmidPoint mutationInferred secondary structure at positionMML160YIplac211No insertMML161pMM25Wild-type GRX5MML163pMM27C60SCoilMML165pMM28C117SHelixMML219pMM76F62SHelixMML221pMM77P58VCoilMML223pMM78K59QCoilMML225pMM79G116VCoilMML273pMM88P58V K59QCoilMML274pMM90P58V F62SCoil, helixMML276pMM92G115VCoilMML322pMM124G61VHelixMML374pMM174F50EBetaMML421pMM202G115ACoilMML423pMM203G115SCoilMML425pMM204G61AHelixMML427pMM205G61SHelixAll strains are isogenic to wild-type S. cerevisiae CML235. The strains listed were derived from MML19 by insertion of the corresponding plasmids. Plasmids (YIplac211 vector or derivatives with wild-type or GRX5 mutants) were integrated at the chromosomal LEU2 locus after transformation with DNA that had been linearized by digestion at the single EcoRV site within the plasmid LEU2 gene. The amino acid replacements introduced in the GRX5 translation product are indicated. The secondary structure elements at the amino acid substitution positions are inferred from the model in Fig. 3. Open table in a new tab All strains are isogenic to wild-type S. cerevisiae CML235. The strains listed were derived from MML19 by insertion of the corresponding plasmids. Plasmids (YIplac211 vector or derivatives with wild-type or GRX5 mutants) were integrated at the chromosomal LEU2 locus after transformation with DNA that had been linearized by digestion at the single EcoRV site within the plasmid LEU2 gene. The amino acid replacements introduced in the GRX5 translation product are indicated. The secondary structure elements at the amino acid substitution positions are inferred from the model in Fig. 3. The following plasmids contain the cloned GRX5 open reading frame (with the type of mutation in the translation product indicated in parentheses) without further upstream or downstream sequences, under the control of the doxycycline-regulatable tetO 7promoter in plasmid pCM190 (29Garı́ E. Piedrafita L. Aldea M. Herrero E. Yeast. 1997; 13: 837-848Crossref PubMed Scopus (504) Google Scholar): pCM319 (wild-type GRX5), pMM176 (F50E), pMM113 (C60S), pMM155 (G61V), pMM127 (G115V), and pMM112 (G116V). These plasmids were then transformed into strain CML276 (30Bellı́ G. Garı́ E. Aldea M. Herrero E. Yeast. 1998; 14: 1127-1138Crossref PubMed Scopus (0) Google Scholar), which carries the doxycycline-inducible tetR′-SSN6 repressor gene, to determine the stability of the Grx5 wild-type protein and the amino acid-substituted derivatives. Point mutations in the GRX5 open reading frame that yielded the different amino acid replacements were constructed by the ExSite method (31Weiner M.P. Costa L. Dieffenbach C.W. Dveksler G.S. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1995: 613-621Google Scholar), using either pMM25 or pCM319 DNA as a template. Oligonucleotides for PCR amplification were designed in such a way that a restriction site that did not alter the translation product was introduced near to the desired point mutation and used as a marker for it. Successful introduction of the mutations was confirmed by DNA sequencing. Cells were usually grown at 30 °C in rich YPD medium. Plasmid-bearing transformants were grown in synthetic complete medium (32Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar) without the selective auxotrophic requirement. Plates of synthetic defined medium (0.67% yeast nitrogen base, 2% glucose, and auxotrophic requirements) were used to test mutant growth. Cells growing exponentially in YPD medium at 30 °C (about 2 × 107 cells/ml) were treated with menadione (10 mm) to determine sensitivity to it. After treatment, 1:5 serial dilutions were made, and drops were spotted onto YPD plates. Growth was recorded after 2 days of incubation at 30 °C. Protein carbonyl levels in crude cell extracts were quantified according to the dinitrophenylhydrazine derivatization method (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). Total iron cell content was determined under reducing conditions, after acid digestion of cells using 3% nitric acid (33Fish W.W. Methods Enzymol. 1988; 158: 357-364Crossref PubMed Scopus (529) Google Scholar). Mean cell volumes were determined in nonfixed cells using a Coulter Z2 counter to calculate cell iron concentration. Exponentially growing cells that overexpressed GRX5 under the control of thetetO 7 promoter were added to doxycycline (20 μg/ml) to interrupt gene expression. At successive times, samples were taken, and total cell extracts were prepared (24Rodrı́guez-Manzaneque M.T. Tamarit J. Bellı́ G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (394) Google Scholar). Western analyses were carried out using a polyclonal antibody prepared againstEscherichia coli-expressed full-length Grx5 protein at a 1:2500 dilution (24Rodrı́guez-Manzaneque M.T. Tamarit J. Bellı́ G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (394) Google Scholar). In each experiment, equal amounts of total cell protein (60 μg) were run in parallel for each sample. The relative level of Grx5 was determined from the intensity of the Grx5 band signal, following quantification with the Lumi-Imager equipment (Roche Molecular Biochemicals) software. Protein structures related to Grx5 were identified by applying the GenThreader fold recognition method (34Jones D.T. J. Mol. Biol. 1999; 287: 797-815Crossref PubMed Scopus (785) Google Scholar). Two structures offering maximal probability of correct match were selected and collected from the Protein Data Bank (Protein Data Bank accession numbers 1KTE and 3GRX). They respectively corresponded to pig liver thioltransferase (7Katti S.K. Robbins A.H. Yang Y. Wells W.W. Protein Sci. 1995; 4: 1998-2005Crossref PubMed Scopus (62) Google Scholar) and E. coliGrx3 glutaredoxin (14Nordstrand K. Aslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar). Different models of Grx5 based on these protein structures were obtained using the Swiss-Model server (35Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9590) Google Scholar, 36Guex N. Diemand A. Peitsch M.C. Trends Biochem. Sci. 1999; 24: 364-367Abstract Full Text Full Text PDF PubMed Google Scholar). Protein structures were analyzed applying the Swiss-PDB Viewer program (35Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9590) Google Scholar). Grx5 amino acid sequence was compared with proteins from the Institute for Chemical Research (Kyoto University, Kyoto, Japan) and Swiss Protein Databases using FASTA analysis provided by the two servers. Multiple sequences were aligned using the ClustalW program (37Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55767) Google Scholar) and the tools provided by the European Bioinformatics Institute. Internal gaps were not eliminated, and the Blosum80 matrix option was used for alignment. Yeast Grx5 has been characterized as a monothiol glutaredoxin-like protein whose amino acid sequence displays extensive homology (particularly at what have been designated its amino-terminal and carboxyl-terminal regions) with a family of proteins whose members are present in all living organisms from bacteria to humans (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar, 25Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The carboxyl-terminal region also has significant homology with classic dithiol glutaredoxins (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). To extend these initial studies, the Institute for Chemical Research and Swiss Protein Databases were searched for proteins with the highest homology with Grx5 (Evalue cutoff, 1 × e −10), using FASTA analysis. We only considered proteins that retained the putative active site CGFS sequence in the amino-terminal region (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar) for comparison. The 35 protein sequences with the highest similarity score with Grx5 were then aligned using the ClustalW program (Fig.1). A putative Grx5 homologue fromCandida albicans, as deduced from the genome sequence of the latter organism, was also included for comparison. Extensions at the amino-terminal and carboxyl-terminal ends (that are present only in some of the family members (see below)) were omitted for the alignment. The existence of two amino-terminal and carboxyl-terminal regions with extensive homology (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar) (separated by a less well-conserved region with a slightly variable length) was confirmed in this extended study. Most multicellular eukaryotic members of the Grx5 family have large amino-terminal extensions. This is also the case for the S. cerevisiae Grx3 and Grx4 glutaredoxins and for one of the two sequences in fission yeast (Fig. 1). This amino-terminal extension includes a highly conserved duplication of the region shown in Fig. 1in the cases of human and rat species and in one of the two mouse species (Q9JLZ2M) (Ref. 25Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar and this study). Interestingly, theArabidopsis thaliana protein Q9ZPH2A (but not other members of the same protein family in this plant species) contains three conserved domains in tandem, but only the most carboxyl-terminal of these is shown in the Fig. 1 alignment. On the other hand, S. cerevisiae Grx5, the C. albicans Grx5 homologous protein, the other fission yeast protein, and all bacterial members of the family have shorter versions of the protein without amino-terminal extensions. The domain shown in the alignments is almost totally coincident with the PICOT homology domain region named after the human Grx5 homologue (25Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 27Witte S. Villalba M., Bi, K. Liu Y. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Our study shows that this domain may be shared by proteins from prokaryotes (both Archaea and bacteria) and eukaryotes. These proteins may have divergent functions and different cellular locations. Grx5 has sequence similarity with dithiol glutaredoxins, mostly at the carboxyl-terminal moiety (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). The three-dimensional structure of a number of dithiol glutaredoxins was already known, from either x-ray crystallography or NMR spectroscopy studies. Structures in the Protein Data Bank were used to construct a three-dimensional model for Grx5. Two protein structures, pig liver thioltransferase (1KTE) (7Katti S.K. Robbins A.H. Yang Y. Wells W.W. Protein Sci. 1995; 4: 1998-2005Crossref PubMed Scopus (62) Google Scholar) and E. coli Grx3 glutaredoxin (3GRX) (14Nordstrand K. Aslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar), yielded useful models for Grx5. Amino acid sequences of these two proteins show 28% and 29% identity to Grx5, respectively. Models based both individually and simultaneously on 1KTEand 3GRX yielded almost identical results for a large, carboxyl-terminal part of the protein, which represented about two-thirds of the sequence. Differences in the amino-terminal part were attributed to the poor quality of the 3GRX-based model because homology of E. coli Grx3 to Grx5 is low in this region. The 1KTEstructure (Fig. 2 A) was therefore finally taken as the basis for the Grx5 model (Fig.2 B), which is considered to provide a valid representation of the Grx5 protein structure. The proposed model shows an αβα fold typical of thiol oxidoreductases and other enzymes from the superfamily (15Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). Four α-helix segments are predicted: amino acids 61–71 (α1), 92–98 (α2), 117–125 (α3), and 129–135 (α4) (Fig. 1, top row). For comparison, Fig. 2 C shows the structure of phage T4 glutaredoxin (1ABA) (6Eklund H. Ingelman M. Söderberg B.-O. Uhlin T. Nordlund P. Nikkola M. Sonnerstam U. Joelsson T. Petratos K. J. Mol. Biol. 1992; 228: 596-618Crossref PubMed Scopus (68) Google Scholar), which is one of the simplest (structurally speaking) representatives of the thioredoxin fold superfamily. The model shows that the similarity between Grx5 and other proteins from the superfamily extends to the amino-terminal moiety of the molecule. Grx5 contains two cysteine residues at positions 60 and 117. The first is part of a conserved sequence common to all members of the family (Ref. 23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar; Fig. 1). It is exposed at the surface of Grx5 between a β-strand and α1 (Fig.3). Cys117 is only present in certain family members (Fig. 1), although many dithiol glutaredoxins also contain a cysteine residue in an equivalent position (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). This cysteine is the first α3 residue and follows two glycine residues that are conserved in all the dithiol and monothiol glutaredoxins (Fig. 3; Ref. 23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). Cys60 and Cys117 were separately substituted by serine residues, and the biological effects of these mutations were studied after reintroducing the respective mutant Grx5 forms into a strain that was devoid of the wild-type version. Initially, two phenotypes were studied that had been shown to be affected in null grx5 mutants (23Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar): sensitivity to oxidative stress induced by menadione, and defective growth in synthetic defined medium. Cells with the C60S mutation in Grx5 behaved like those without the protein, whereas those with the C117S change did not differ with respect to wild-type cells (Fig. 4 B). Therefore, the absence of Cys60 annuls the biological activity of Grx5, whereas Cys117 is not essential for it. These results demonstrate that Cys60 is the active residue for the oxidoreductase reaction and confirm observations for two dithiol glutaredoxins, pig liver thioltransferase (4Yang Y. Wells W.W. J. Biol. Chem. 1991; 266: 12759-12765Abstract Full Text PDF PubMed Google Scholar) and E. coliGrx3 (14Nordstrand K. Aslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar), where the cysteine residues at equiva" @default.
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• W2062917114 title "Structure-Function Analysis of Yeast Grx5 Monothiol Glutaredoxin Defines Essential Amino Acids for the Function of the Protein" @default.
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