Molecular Mapping of Functionalities in the Solution Structure of Reduced Grx4, a Monothiol Glutaredoxin from Escherichia coli

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 of protein to of the active site of reductase (reviewed in 1Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (542) Google and A. Proc. Natl. Acad. Sci. U. S. A. 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Full Text Full Text PDF PubMed Scopus (112) Google Scholar). substrate found of the which is in oxidative stress S. M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar), two monothiol glutaredoxin but their are The monothiol glutaredoxin in E. glutaredoxin recently as essential in a Campbell E. A. M. J. PubMed Scopus Google Scholar). with to a of the have which that Grx4 are 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 is well and with properties as 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 classic glutaredoxin activity small and known substrates glutaredoxins thioredoxins such as HED, and reductase are essential glutaredoxin 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 from the classic glutaredoxins by a substrate thioredoxin reductase and glutathione 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 can be and is with high by 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 is but with high substrate both dithiol glutaredoxins the activity of the Grx4 monothiol in E. as in yeast M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar), and the E. coli Grx4 monothiol is essential cell and the close between the monothiol and dithiol glutaredoxins, the of the monothiol glutaredoxins the of the monothiol glutaredoxins, their lack of activity in the glutaredoxin 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 The by the monothiol glutaredoxins that structural of monothiol glutaredoxins using a dithiol J. J. M.T. Ros J. Herrero E. J. Biol. 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PubMed Scopus Google Scholar). in a Grx4 a fold, which consists of a flanked by α-helices secondary structure identified and in the of the NMR together with form a structure as a classic a conserved in the of the thioredoxin fold, but between and are the the formation of a and are on the of the β-sheet and are to and are on the of the where and are and are in but are structurally by most likely by the conserved between the The of is to the between and through a and which be in the of are found in the core, are well are exposed and in exposed which is strictly conserved the monothiol and residues and in the exposed of and A set of residues with of the of Grx4 all of of and the and the conserved glutaredoxin Holmgren A. Berndt J. Mol. Biol. 1999; PubMed Scopus (112) Google and and to the and are but form part of secondary structure The suggested active site of Grx4 is on the to and in the structure are both the active site and which be to conformational to the exposed of the of the active site are as suggested by the by which is in close The from the of and the absence of between residues and A close to the active site is found in most and is to a in and Vlamis-Gardikas A. Holmgren A. Wright P.E. Dyson H.J. J. Mol. Biol. PubMed Scopus Google Scholar). a between and and a linking the of with be in the of the CXFX of the a the is conserved in all monothiol glutaredoxins The surface of Grx4 shows two hydrophobic and where the of the β-sheet from the protein core, but with a that is to the by a of and are a and the solution to NMR The and of that be of functional protein Structural of in the to the conserved in the active site which is the of the monothiol glutaredoxin a set of conserved residues in the protein suggest a conserved a common structural The as well as the are well conserved in both the monothiol and dithiol glutaredoxin the in which is close to the active is strictly in the monothiol glutaredoxins, residues and are found in the dithiol the monothiol glutaredoxins conserved significant of the be with is as A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. 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Grx4 is structurally to the glutaredoxin of from two and the glutaredoxin-like protein The thioredoxin proteins identified as closely structurally related to most likely the thioredoxins have found in Grx4 glutaredoxins. in Grx4 a structure of a monothiol glutaredoxin is a of the conserved surface with the glutathione binding of Holmgren A. Berndt J. Mol. Biol. 1999; PubMed Scopus (112) Google and Holmgren A. PubMed Scopus Google that Grx4 a of glutathione binding that of Grx3. The glutathione binding residues of are in Grx4 the of the two proteins the differences in and between the two the surface of the conserved glutathione binding residues is and forms a surface of and and small is in the is exposed and in glutathione the residues in Grx4 are and the exposed which is strictly conserved in monothiol glutaredoxins. the surface of E. coli and is more and a more The of the glutathione binding that monothiol glutaredoxins a of glutathione and that in ligand binding. Structural of MALDI in Grx4 on of MALDI which the activity of thioredoxin reductase on oxidized Grx4 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), structural in the cycle of Grx4 of which is in the monothiol experiments the of disulfide formation on glutathionylation 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). thioredoxin reductase to reduce Grx4 but to by to in and Grx4 with to the formation of 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). in the of the present Grx4 the MALDI suggest structural on glutathionylation and disulfide formation is to be to as is based on the of the binding site. The two and must be in oxidized Grx4. the reduced and are on the of and with a of The formation of a disulfide on oxidative glutathionylation must be related to a structural by of both and by of that the to The by thioredoxin reductase of the disulfide but the and of 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). of Grx4 in on all the structure of is close to the suggested and well be on the from of is likely to of on The the function of the monothiol glutaredoxins a the solution structure of Grx4. that monothiol glutaredoxins are essential in both E. coli and yeast A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. 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PubMed Scopus Google Scholar), their main activity is The structure of Grx4 a more of the of conserved in the monothiol family is from is that the of Grx4 is in all monothiol glutaredoxin identified to thus a closely conserved The structure is closely related to the well known fold, structure such as a conserved and a close to the active site. are by the properties of Grx4 with the dithiol in experiments A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. 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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). MALDI together with the structure of reduced Grx4 suggest that significant structural are in of monothiol Grx4 of secondary structure to the disulfide linking between and which are on of and in the reduced a structure of the oxidized form of Grx4 is to is be that small in the of to and to disulfide formation between and The of two of which is A of the between and is to to the The and are conserved in the of all with a single monothiol glutaredoxin in conserved The of structural on Grx4 oxidation with of the and E. coli thioredoxins as well as E. coli where the between oxidized and reduced forms are subtle and Holmgren A. Berndt J. Mol. Biol. 1999; PubMed Scopus (112) Google Scholar, A. Structure. Full Text Full Text PDF PubMed Scopus Google Scholar). disulfide formation on glutathione binding to the E. coli and monothiol glutaredoxins and the well form disulfide between residues to and in Grx4 be that oxidation of as in is a where of disulfide disulfide two closely is by formation of a disulfide on the M. J. Chem. Scopus Google Scholar). the structural change on glutathione binding that formation is likely to in monothiol glutaredoxins. the monothiol glutaredoxins with the glutathionylation by that the disulfide of E. coli Grx4 be a substrate of TrxR, as between the thioredoxin and glutaredoxin 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), of the as active site of Grx4. is that the in the between the on the surface of and in Grx4 are well The structure of Grx4 thus both from a and the main active of the monothiol glutaredoxins be in the CXFX the suggested of structural change in Grx4 on oxidation that which dithiol glutaredoxins, have that in The of the a is to be disulfide by the of that the in formation M. U. Dyson H.J. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). in the thioredoxin a in structure from to with structural the thioredoxin E. J. Mol. Biol. PubMed Scopus Google Scholar). well be part of the of Grx4 the significant hydrophobic in the Grx4 and the limited the monothiol glutaredoxins with in to that in the CXFX a structural change on glutathionylation disulfide formation with in such be in E. coli thioredoxin and thioredoxin reductase found to with and in a to that of that are in protein and protein stress A. Holmgren A. J. PubMed Scopus Google Scholar). The present the solution structure of a monothiol glutaredoxin, with high to of protein present in living and They have to be in the formation of in proteins and to in M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar), and signal by the activity of protein S. M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The present to the of monothiol in particular with to glutathionylation and disulfide form the the of monothiol glutaredoxins in the cellular defense against oxidative stress and monothiol glutaredoxins to and and of protein family be essential to their in the of the Andrésen is of the MALDI and are NMR with