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• W2008554228 abstract "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 α-β 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. 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 α-β 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. Glutaredoxins are ubiquitous proteins found in most living organisms, from prokaryotes to humans. They employ glutathione (GSH) 1The abbreviations used are: GSH, reduced glutathione; Grx, glutaredoxin; HED, β-hydroxyethyl disulfide; MALDI, matrix-assisted laser desorption/ionization mass spectrometry; Trx, thioredoxin; TrxR, thioredoxin reductase; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; r.m.s.d., root mean square deviation; GST, glutathione S-transferase. to catalyze redox-dependent cellular functions, such as transcription and biosynthesis regulation, signal transduction, cell cycle control, and protection against oxidative stress (reviewed in Ref. 1Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (542) Google Scholar). The glutaredoxin family of proteins is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism in a coupled system with NADPH and thioredoxin reductase (TrxR) (2Vlamis-Gardikas A. Holmgren A. Methods Enzymol. 2002; 347: 286-296Crossref PubMed Scopus (112) Google Scholar). The well conserved thioredoxin fold consists of a five-stranded β-sheet flanked by four α-helices (3Holmgren A. Soderberg B.O. Eklund H. Branden C.I. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2305-2309Crossref PubMed Scopus (368) Google Scholar), where a hydrophobic surface close to the active site (CGPC) mediates substrate binding. A localized conformational change accompanies oxidation of the dithiol form to the disulfide form (4Jeng M.F. Campbell A.P. Begley T. Holmgren A. Case D.A. Wright P.E. Dyson H.J. Structure. 1994; 2: 853-868Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The glutaredoxins can be divided into two subfamilies according to their active sites: the classic dithiols, with CPXC as the active site and the more recently identified monothiol glutaredoxins, having CXFX as their suggested active site (1Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (542) Google Scholar, 5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar, 6Rodriguez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). Careful characterization of dithiol glutaredoxins shows that their main function is the reduction of functionally important protein disulfides, leading to activation and/or inactivation of biological activity. Glutaredoxin targets include the active site of ribonucleotide reductase (reviewed in Refs. 1Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (542) Google Scholar and 7Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2275-2279Crossref PubMed Scopus (364) Google Scholar), which is essential for DNA synthesis, a disulfide in 3′-phosphoadenylyl sulfate reductase (8Tsang M.L. Schiff J.A. J. Bacteriol. 1978; 134: 131-138Crossref PubMed Google Scholar), the key enzyme in the reduction of sulfate to sulfite (sulfur assimilation), and the mixed disulfide between arsenate reductase and glutathione forming upon reduction of arsenate to arsenite ions (9Shi J. Vlamis-Gardikas A. Åslund F. Holmgren A. Rosen B.P. J. Biol. Chem. 1999; 274: 36039-36042Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Glutaredoxins can also maintain the activity of redox-sensitive proteins by deglutathionylating cysteines that may form mixed disulfides with glutathione upon oxidative conditions resulting in loss of biological activity (10Sodano P. Xia T.H. Bushweller J.H. Bjornberg O. Holmgren A. Billeter M. Wüthrich K. J. Mol. Biol. 1991; 221: 1311-1324Crossref PubMed Scopus (91) Google Scholar, 11Lillig C.H. Potamitou A. Schwenn J.D. Vlamis-Gardikas A. Holmgren A. J. Biol. Chem. 2003; 278: 22325-22330Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Several three-dimensional structures of dithiol glutaredoxins have been determined (10Sodano P. Xia T.H. Bushweller J.H. Bjornberg O. Holmgren A. Billeter M. Wüthrich K. J. Mol. Biol. 1991; 221: 1311-1324Crossref PubMed Scopus (91) Google Scholar, 12Eklund H. Ingelman M. Soderberg B.O. Uhlin T. Nordlund P. Nikkola M. Sonnerstam U. Joelson T. Petratos K. J. Mol. Biol. 1992; 228: 596-618Crossref PubMed Scopus (68) Google Scholar, 13Åslund 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, 14Sun C. Berardi M.J. Bushweller J.H. J. Mol. Biol. 1998; 280: 687-701Crossref PubMed Scopus (79) Google Scholar, 15Xia B. Vlamis-Gardikas A. Holmgren A. Wright P.E. Dyson H.J. J. Mol. Biol. 2001; 310: 907-918Crossref PubMed Scopus (69) Google Scholar). All of these glutaredoxins contain a thioredoxin-like α-β fold where the CPXC motif extends from the core in a loop-like structure, and the central β-sheet is composed of four strands compared with the five strands in thioredoxins (reviewed in Ref. 1Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (542) Google Scholar). Glutathione binding sites have been characterized in atomic detail for human Grx1, Escherichia coli Grx1, and E. coli Grx3, all of which involve covalent linkage of glutathione to the N-terminal cysteine in the active site loop (16Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (131) Google Scholar, 17Nordstrand K. Åslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar, 18Bushweller J.H. Åslund F. Wüthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar). Furthermore, dithiol glutaredoxins share the common property of reducing small molecular weight disulfides with GSH, as shown in the HED assay (17Nordstrand K. Åslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar, 18Bushweller J.H. Åslund F. Wüthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar). Recent studies indicate that, despite significant sequence similarities, monothiol glutaredoxin functionality is clearly distinct from that of dithiol glutaredoxins. The monothiol glutaredoxin motif occurs in multidomain arrangements in multicellular eukaryotic species, whereas in lower organisms, only single domain monothiol glutaredoxins have been conserved (19Belli G. Polaina J. Tamarit J. De La Torre M.A. Rodriguez-Manzaneque M.T. Ros J. Herrero E. J. Biol. Chem. 2002; 22: 22Google Scholar, 20Witte 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 monothiol Grx5 from yeast is localized in the mitochondria and performs thioloxidoreductase activity through deglutathionylation of carbonic anhydrase III (21Rodriguez-Manzaneque M.T. Tamarit J. Belli G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (395) Google Scholar, 22Rahlfs S. Fischer M. Becker K. J. Biol. Chem. 2001; 276: 37133-37140Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 23Tamarit J. Belli G. Cabiscol E. Herrero E. Ros J. J. Biol. Chem. 2003; 278: 25745-25751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Yeast Grx5 is also required for the activity of Fe-S cluster enzymes (21Rodriguez-Manzaneque M.T. Tamarit J. Belli G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (395) Google Scholar), which suggests a role in protecting the cell against oxidative stress by regulating the activity of Fe-S cluster proteins. A Plasmodium falciparum multidomain protein with a monothiol glutaredoxin domain reduces insulin dimers (22Rahlfs S. Fischer M. Becker K. J. Biol. Chem. 2001; 276: 37133-37140Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Neither of these monothiol glutaredoxins reduce small molecular weight disulfides with GSH, and thus appear to lack the classic glutaredoxin activity (22Rahlfs S. Fischer M. Becker K. J. Biol. Chem. 2001; 276: 37133-37140Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 23Tamarit J. Belli G. Cabiscol E. Herrero E. Ros J. J. Biol. Chem. 2003; 278: 25745-25751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Furthermore, no substrate has been found for any of these proteins. Among eukaryotic proteins, the human PICOT protein, which is important in oxidative stress response (20Witte 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, 24Babichev Y. Isakov N. Adv. Exp. Med. Biol. 2001; 495: 41-45Crossref PubMed Scopus (25) Google Scholar), contains two monothiol glutaredoxin domains, but their specific functions are still unknown. The only monothiol glutaredoxin in E. coli, glutaredoxin 4 (Grx4), was recently classified as being essential in a gene footprinting study (25Gerdes S.Y. Scholle M.D. Campbell J.W. Balazsi G. Ravasz E. Daugherty M.D. Somera A.L. Kyrpides N.C. Anderson I. Gelfand M.S. Bhattacharya A. Kapatral V. D'Souza M. Baev M.V. Grechkin Y. Mseeh F. Fonstein M.Y. Overbeek R. Barabasi A.L. Oltvai Z.N. Osterman A.L. J. Bacteriol. 2003; 185: 5673-5684Crossref PubMed Scopus (594) Google Scholar). In agreement with this, several attempts to produce a knock-out of the gene have failed, which indicates that cells lacking Grx4 are not viable (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar). Recombinantly expressed Grx4 (115 amino acids) is well folded and thermally stable, with similar biophysical properties as Grx1 (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar). Grx4 lacks classic glutaredoxin activity toward small disulfides, and other known substrates for glutaredoxins or thioredoxins such as HED, insulin, and 3′-phosphoadenylyl sulfate reductase are not targets for this essential glutaredoxin (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar). Grx4 also differs from the classic glutaredoxins by being a substrate for thioredoxin reductase and not for glutathione (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar). However, Grx4 can be glutathionylated and is deglutathionylated with high selectivity by Grx1 (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar). Thus, Grx4 is redoxactive, but with high substrate specificity. This may explain both why normal dithiol glutaredoxins cannot compensate for the activity of the Grx4 monothiol species in E. coli, as was observed for Grx5 in yeast (6Rodriguez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar), and why the E. coli Grx4 monothiol is essential for cell growth and viability. Despite the close sequence homology between the monothiol and dithiol glutaredoxins, the functionality of the monothiol glutaredoxins remains elusive. In particular, the essentiality of the monothiol glutaredoxins, despite their lack of observable activity in the standard glutaredoxin assays (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar), is intriguing. The distinct functionalities displayed by the monothiol glutaredoxins suggests that structural modeling of monothiol glutaredoxins using a dithiol template (19Belli G. Polaina J. Tamarit J. De La Torre M.A. Rodriguez-Manzaneque M.T. Ros J. Herrero E. J. Biol. Chem. 2002; 22: 22Google Scholar) will not adequately predict functionalities of this novel motif. To further characterize the properties of essential monothiol glutaredoxins, we have employed high resolution NMR to determine the three-dimensional structure of a monothiol glutaredoxin, the reduced E. coli Grx4, which is described in this work. Structural and functional implications of the monothiol fold are discussed and analyzed with particular emphasis on substrate specificity and binding events. Protein Expression and Sample Preparation—Unlabeled Grx4 was overexpressed in E. coli strain BL21(DE3)grxA–grxB–grxC– using the T7 polymerase/promoter system (pET-15b), as described previously (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar). Isotopically labeled Grx4 was produced in rich growth OD2 media from Silantes, containing primary amino acids, some low molecular weight oligopeptides, and almost no carbohydrates. Cells from an overnight culture of unlabeled Silantes OD2 media were spun down, and the pellet was resuspended in 15N- or 13C/15N-labeled Silantes OD2 media containing 100 μg/ml ampicillin. Both unlabeled and labeled protein samples were purified as described earlier (5Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; (April 15, 10.1074/jbc.M500678200)Google Scholar) and concentrated to 0.75 mm in 125 mm KCl and 5 mm PO4, pH 6.5. NMR Measurements and Spectral Evaluation—NMR experiments were performed at 28 °C, using Bruker DMX 600 and Varian Unity INOVA 800 NMR spectrometers. Unlabeled, 15N-labeled, and 13C/15N-labeled proteins were employed. The requirement for KCl in the purification of Grx4 was significant for obtaining spectra with narrow linewidths at near-millimolar protein concentrations. Sequence-specific resonance assignment was obtained from analysis of spin systems and sequential patterns in 15N-13C-HNCA, 15N-HSQC-NOESY, and 15N-HSQC-TOCSY spectra in H2O, together with analysis of homonuclear NOESY (mix 40 ms, 120 ms), TOCSY (mix 100 ms), and COSY spectra in H2O, and of double-quantum filtered COSY, TOCSY (mix 40 ms) and NOESY spectra in D2O. NOEs were obtained from NOESY spectra in H2O and D2O recorded with 40-ms mixing time at 800 MHz. Coupling constants were derived from HSQC and NOESY cross-peak line-fitting (26Szyperski T. Guentert P. Otting G. Wuethrich K. J. Magn. Reson. 1992; 99: 552-560Google Scholar) and from the comparison of signal intensities in a pair of constant-time 15N,1H-HMQC spectra recorded with and without decoupling of the JHNHα coupling (27Ponstingl H. Otting G. J. Biomol. NMR. 1998; 12: 319-324Crossref PubMed Scopus (25) Google Scholar). NMR data were processed with the program PROSA (28Guntert P. Dotsch V. Wider G. Wüthrich K. J. Biomol. NMR. 1992; 2: 619-629Crossref Scopus (280) Google Scholar). All spectra were analyzed, and peaks were integrated with the program XEASY (29Bartels C. Xia T. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 5: 1-10Crossref PubMed Scopus (1607) Google Scholar). Structure Calculation—The program ARIA1.2 (Ambiguous Restraints for Interactive Assignment) (30Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (333) Google Scholar), as an extension of CNS1.1, was used to compute the solution structure of Grx4. NOE cross-peaks from the two-dimensional NOESY and three-dimensional NOESY spectra and chemical shift assignments were used as input to ARIA together with 77 backbone ϕ angles, which were constrained to –60° ± 20°, –120° ± 40°, and –120° ± 20° for small (<5.5 Hz), large (8 ± 1 Hz), and very large (>9 Hz) 3JHNHα couplings, respectively. Floating chirality assignment was used for all methylene and isopropyl groups with separate chemical shifts. The experimentally determined distance and dihedral-angle restraints were applied in a simulated annealing protocol using the program CNS, where the starting structure consisted of an extended structure with random side-chain conformations. Optimization of the structure calculation protocol for ambiguous distance restraints and violation analysis was performed as previously described (31Nilges M. O'Donoghue S.I. Progr. NMR Spectr. 1998; 32: 107-139Abstract Full Text PDF Scopus (224) Google Scholar). The NOEs were calibrated and largely automatically assigned during the structure calculation by ARIA. The NOEs assigned after eight cycles of structure calculation were subjected to a process of manual editing by a careful re-examination of the spectra to improve the quality of the data set used for structure calculations. The new list of 2703 edited NOEs was used as input for a new set of calculations. Finally, backbone torsion angle restraints derived from chemical shifts using TALOS (32Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar) were added to further improve the already converged unique structure. The dihedral angle restraints were taken to be ±2 S.D. values or at least ± 20° from the average values predicted by TALOS. A short molecular dynamics simulation in a thin layer of explicit water was used to refine the final structure ensemble (33Linge J.P. Williams M.A. Spronk C. Bonvin A. Nilges M. Proteins Struct. Funct. Genet. 2003; 50: 496-506Crossref PubMed Scopus (544) Google Scholar). The resulting 20 energy-minimized conformations were selected to represent the NMR structure of Grx4. Structure Evaluation—The quality of the structures were evaluated using the PROCHECK (34Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4470) Google Scholar) and WHATCHECK (35Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1816) Google Scholar) softwares. The structures were displayed and analyzed using the MOLMOL (36Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar) program, which was also, together with PROCHECK, used to calculate root mean square deviation (r.m.s.d.). WHATIF was used to identify hydrogen bonds from structural criteria as well as tentative hydrogen bonds made possible with minor side-chain adjustments (Optimal Hydrogen Bonding Network (35Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1816) Google Scholar)). The three-dimensional structural similarity was assessed using the programs DALI (37Holm L. Sander C. Nucleic Acids Res. 1999; 27: 244-247Crossref PubMed Scopus (177) Google Scholar) and VAST (38Gibrat J.F. Madej T. Bryant S.H. Curr. Opin. Struct. Biol. 1996; 6: 377-385Crossref PubMed Scopus (890) Google Scholar, 39Madej T. Gibrat J.F. Bryant S.H. Proteins. 1995; 23: 356-369Crossref PubMed Scopus (374) Google Scholar), and the hits were evaluated using r.m.s.d. divided by the number of aligned residues (r.m.s.d./Nalign) as described elsewhere (40Sierk M.L. Pearson W.R. Protein Sci. 2004; 13: 773-785Crossref PubMed Scopus (98) Google Scholar). The program Consurf (41Glaser F. Pupko T. Paz I. Bell R.E. Bechor-Shental D. Martz E. Ben-Tal N. Bioinformatics. 2003; 19: 163-164Crossref PubMed Scopus (951) Google Scholar) was employed to identify functionally important regions on the surface of the protein, based on the phylogenetic relations between close sequence homologues to Grx4. Protein Data Bank Accession Number—The coordinates of the 20 energy minimized conformers of Grx4 were deposited in the RCSB Protein Data Bank, with the accession number 1YKA. The Solution Structure of Grx4—The high resolution structure of Grx4 was calculated from experimental constraints (Table I) by the structure calculation program ARIA (30Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (333) Google Scholar). Grx4 was completely assigned except for the 4 N-terminal residues, for which no amide proton resonance was observed, and Pro25, where no resonances were observed, most likely due to broadening by exchange in cis-trans isomerization. The 10 lowest energy structures were chosen to represent the NMR structure of Grx4 (Fig. 1A). An evaluation of the structure statistics of the ensemble (Table I) shows a well defined core domain, where the r.m.s.d. values for the regular secondary structure and the backbone heavy atoms of the ensemble were 0.49 and 0.58 Å, respectively. Within this core domain, which comprises residues 5–115, 84% of the residues are well distributed in the most favored region in the Ramachandran plot. The N-terminal 4 residues were unassigned due to rapid amide exchange and are therefore not included in the structure statistics. Long and medium range NOEs are well distributed over the entire protein (see Supplemental Material).Table IStructural statistics of Grx4Structural characteristics for the 10 lowest energy structures of Grx4Distance restraintsIntraresidue1034Sequential607Medium range, i — j ≤ 5374Long range, i — j > 5688All unambiguous2491All ambiguous212Dihedral angles restraints3JHNHa-derived ϕ77TALOS-derived ϕ, φ82, 82r.m.s.d. from idealityaAverage values.Bonds (Å)0.0052 ± 0.0001Angles (°)0.75 ± 0.02Improper ones (°)1.80 ± 0.11r.m.s.d. from experimental dataaAverage values.Unambiguous NOEs (Å)0.048 ± 0.015Ambiguous NOEs (Å)0.056 ± 0.004All NOEs (Å)0.049 ± 0.014Torsion angles constraints (°)2.08 ± 0.22Ensemble r.m.s.d. (Å)Secondary structure (backbone)bAverage r.m.s.d. with respect to the mean calculated with MOL-MOL (36).0.56 ± 0.11Secondary structure (heavy)bAverage r.m.s.d. with respect to the mean calculated with MOL-MOL (36).1.10 ± 0.10Backbone (residues 5-115)bAverage r.m.s.d. with respect to the mean calculated with MOL-MOL (36).0.69 ± 0.09Heavy atoms (residues 5-115)bAverage r.m.s.d. with respect to the mean calculated with MOL-MOL (36).1.24 ± 0.08Ramachandran plot appearance (residue 5-115)cCalculated with PROCHECK-NMR (34).Most favored regions (%)83.9Additionally allowed regions (%)12.7Generously allowed regions (%)3.4Disallowed regions (%)0.0a Average values.b Average r.m.s.d. with respect to the mean calculated with MOL-MOL (36Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar).c Calculated with PROCHECK-NMR (34Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4470) Google Scholar). Open table in a new tab Grx4 has a glutaredoxin/thioredoxin-like fold, which consists of a four-stranded β-sheet, flanked by five α-helices (Fig. 1A). Regular secondary structure elements were identified for residue 5–14 (α1), 17–21 (β1), 33–43 (α2), 48–51 (β2), 56–66 (α3), 73–76 (β3), 79–80 (β4), 84–93 (α4), and 95–107 (α5) in the majority of the NMR conformers. Residues 81–82, together with residue 74, form a structure almost definable as a classic β-bulge, a conserved feature in the corresponding position of the thioredoxin fold, but distances between 74O and 82NH are slightly above the threshold formally required for the formation of a hydrogen bond. Helices α1 and α3 are located on the same side of the β-sheet and are oriented orthogonally to each other. Helices α2, α4, and α5 are located on the opposite side of the β-sheet, where α2 and α4 are essentially parallel. Helices α4 and α5 are almost continuous in sequence but are structurally tilted by 90°, most likely facilitated by the conserved Gly94 interspaced between the helices. The C terminus of α5 is connected to the short loop between β3 and β4 through a hydrogen bond involving Tyr107 Oη and Asp77 Oδ1, which may be important in defining the orientation of α5. Well ordered side chains (r.m.s.d. < 1) are predominantly found in the core, or are partly buried (Fig. 1B). Seven well ordered side chains are exposed (>30%), including Gln54 and Pro56, in an exposed loop preceding α3, Pro69, which is strictly conserved within the monothiol family, and residues 38, 42, 89, and 92, in the exposed C-terminal parts of α2 and α4 (Fig. 1B). A set of 26 residues with side-chain solvent exposures of <10% defines the core of Grx4 (Fig. 2A). This core includes all of β1, one face of α2, β3 and the preceding loop, and the conserved GG glutaredoxin signature sequence (17Nordstrand K. Åslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar) (residues 82–83). Residues Ile8, Ile12, Leu62, Pro63, Val87, Leu96, Ile100, Thr103, and Thr104 connect α1, α3, α4, and α5 to the solvent-excluded core. Residues Leu44, Ile52, and Asp113 are buried but do not form part of any secondary structure element. The suggested active site of Grx4 (Cys30–Ser33) is located on the molecular surface, N-terminal to α2, and appears partially disordered in the structure ensemble. Indeed, NOEs are scarce for both the active site and several preceding residues, which may be due to conformational exchange and/or to the exposed nature of the loop. However, parts of the active site region are rigid, as suggested by the cis-conformation adopted by Pro72, which is in close spatial proximity. The cis-peptide conformation was deduced from the presence of sequential dαα and the absence of sequential dαβ NOE between residues 71 and 72. A corresponding cis-Pro close to the active site is found in most glutaredoxins/thioredoxins and is thought to play a role in folding and redox dynamics (15Xia B. Vlamis-Gardikas A. Holmgren A. Wright P.E. Dyson H.J. J. Mol. Biol. 2001; 310: 907-918Crossref PubMed Scopus (69) Google Scholar). Furthermore, a" @default.
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• W2008554228 title "Molecular Mapping of Functionalities in the Solution Structure of Reduced Grx4, a Monothiol Glutaredoxin from Escherichia coli" @default.
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