Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750 -2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3,5-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like ␣-␤ fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.

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Although apparently functionally unrelated, intracellular TRAFs and extracellular meprins share a region with conserved meprin and traf homology, MATH 1 . Both TRAFs and meprins require subunit assembly for function. By structural analysis of the sequences, we provide an explanation of how meprins, which form tetramers, and TRAF molecules, which form trimers, can share homology. Our analysis suggests it is highly likely that the same oligomerization surface is used. The analysis has implications for the widely distributed group of proteins containing MATH domains. Both TRAFs and meprins require subunit assembly for function. Trimerization mainly by the TRAF-N trimeric coiled-coil motif and by TRAF-C domain interactions appears crucial for establishing appropriate connections to form signalling complexes with TNF receptor-1 and related death receptors [7^10]. Meprins A and B form membranebound tetrameric complexes, dimers of heterodimers, through interactions between MAM domains N-terminal to the MATH motif [11,12], and the tetrameric state was also observed within the secreted meprin A homo-oligomer [13]. A meprin mutant incapable of forming the tetrameric state lost activity toward proteins, but could still hydrolyze peptide substrates [12].Residues in meprins A and B that are highly similar with TRAF-C domains are located throughout the L-barrel, including the eighth strand which was only recently included in the MATH motif [14] (Fig.

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ErratumErratum to: The new MATH: homology suggests shared binding surfaces in meprin tetramers and TRAF trimers (FEBS 26538) [FEBS Letters 530 (2002) 1^3]