Structural Basis of Redox-coupled Protein Substrate Selection by the Cytochrome c Biosynthesis Protein ResA
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m402823200
ISSN1083-351X
AutoresAllister Crow, Richard M. Acheson, Nick E. Le Brun, Arthur Oubrie,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoPost-translational maturation of cytochromes c involves the covalent attachment of heme to the Cys-Xxx-Xxx-Cys-His motif of the apo-cytochrome. For this process, the two cysteines of the motif must be in the reduced state. In bacteria, this is achieved by dedicated, membrane-bound thiol-disulfide oxidoreductases with a high reducing power, which are essential components of cytochrome c maturation systems and are also linked to cellular disulfide-bond formation machineries. Here we report high-resolution structures of oxidized and reduced states of a soluble, functional domain of one such oxidoreductase, ResA, from Bacillus subtilis. The structures elucidate the structural basis of the protein's high reducing power and reveal the largest redox-coupled conformational changes observed to date in any thioredoxin-like protein. These redox-coupled changes alter the protein surface and illustrate how the redox state of ResA predetermines to which substrate it binds. Furthermore, a polar cavity, present only in the reduced state, may confer specificity to recognize apo-cytochrome c. The described features of ResA are likely to be general for bacterial cytochrome c maturation systems. Post-translational maturation of cytochromes c involves the covalent attachment of heme to the Cys-Xxx-Xxx-Cys-His motif of the apo-cytochrome. For this process, the two cysteines of the motif must be in the reduced state. In bacteria, this is achieved by dedicated, membrane-bound thiol-disulfide oxidoreductases with a high reducing power, which are essential components of cytochrome c maturation systems and are also linked to cellular disulfide-bond formation machineries. Here we report high-resolution structures of oxidized and reduced states of a soluble, functional domain of one such oxidoreductase, ResA, from Bacillus subtilis. The structures elucidate the structural basis of the protein's high reducing power and reveal the largest redox-coupled conformational changes observed to date in any thioredoxin-like protein. These redox-coupled changes alter the protein surface and illustrate how the redox state of ResA predetermines to which substrate it binds. Furthermore, a polar cavity, present only in the reduced state, may confer specificity to recognize apo-cytochrome c. The described features of ResA are likely to be general for bacterial cytochrome c maturation systems. c-Type cytochromes are crucial for respiration in many biological species (1Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). They have considerable structural and functional flexibility. Perhaps the best known are the mono-heme proteins that shuttle electrons between respiratory membrane-bound, proton-translocating protein complexes, i.e. from the cytochrome bc1 complex to cytochrome c oxidase. Many bacteria contain multi-heme c-type cytochromes in which the hemes have electron transfer and catalytic activities, e.g. cytochrome c nitrite reductase (2Einsle O. Messerschmidt A. Stach P. Bourenkov G.P. Bartunik H.D. Huber R. Kroneck P.M. Nature. 1999; 400: 476-480Crossref PubMed Scopus (265) Google Scholar, 3Bamford V.A. Angove H.C. Seward H.E. Thomson A.J. Cole J.A. Butt J.N. Hemmings A.M. Richardson D.J. Biochemistry. 2002; 41: 2921-2931Crossref PubMed Scopus (134) Google Scholar). Many bacteria also contain enzymes with heme c and at least one other redox cofactor, e.g. hemocytochromes c (4Fülöp V. Moir J.W. Ferguson S.J. Hajdu J. Cell. 1995; 81: 369-377Abstract Full Text PDF PubMed Scopus (251) Google Scholar), flavocytochromes c (5Bamford V. Dobbin P.S. 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Recent years have seen great advances in our understanding of the structure and function of a diverse range of c-type cytochromes (10Pettigrew G.W. Moore G.R. Cytochromes c: Biological Aspects. Springer-Verlag, Berlin1987Crossref Google Scholar, 11Moore G.R. Pettigrew G.W. Cytochromes c: Evolutionary, Structural and Physicochemical Aspects. Springer-Verlag, Berlin1990Crossref Google Scholar, 12Scott R.A. Mauk A.G. Cytochrome c: A Multidisciplinary Approach. University Science Books, 1996Google Scholar) and their (interaction with) redox partners (13Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1967) Google Scholar, 14Wilmanns M. Lappalainen P. Kelly M. Sauer-Eriksson E. Saraste M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11955-11959Crossref PubMed Scopus (175) Google Scholar, 15Xia D. Yu C.A. Kim H. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (867) Google Scholar, 16Iwata S. 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Largely on the basis of genetic information, three markedly different CCM systems have been identified. Systems I and II occur predominantly in bacteria, whereas system III is found exclusively in eukaryotes (1Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 19Le Brun N.E. Bengtsson J. Hederstedt L. Mol. Microbiol. 2000; 36: 638-650Crossref PubMed Scopus (79) Google Scholar, 20Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (101) Google Scholar).The type I system of Escherichia coli (Fig. 1A) comprises the membrane-bound proteins CcmABCDEFGH. CcmA, CcmB, and CcmC form an ABC-type transporter, the function of which is unknown (20Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (101) Google Scholar, 21Cook G.M. 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Reduction of the cysteinyls of the Cys-Xxx-Xxx-Cys-His motif prior to heme insertion is accomplished by CcmG and CcmH, but it is not clear whether the electrons flow from CcmG by means of CcmH, or from CcmH via CcmG, to the apo-cytochrome (27Fabianek R.A. Hofer T. Thöny-Meyer L. Arch. Microbiol. 1999; 171: 92-100Crossref PubMed Scopus (79) Google Scholar, 28Reid E. Cole J. Eaves D.J. Biochem. J. 2001; 355: 51-58Crossref PubMed Scopus (60) Google Scholar). In addition to these CCM-specific proteins, DsbA, which catalyzes the formation of disulfide bonds in the periplasm, and DsbB, which releases the electrons obtained from DsbA to the respiratory system by means of the quinol pool, have been reported to be important for formation of a disulfide bond in the apo-cytochrome prior to CCM (29Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 30Bardwell J.C. Lee J.O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. 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Chem. 2003; 278: 17852-17858Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The type II system of Bacillus subtilis (Fig. 1B) comprises the ResA, ResB, and ResC proteins (19Le Brun N.E. Bengtsson J. Hederstedt L. Mol. Microbiol. 2000; 36: 638-650Crossref PubMed Scopus (79) Google Scholar, 39Erlendsson L.S. Acheson R.M. Hederstedt L. Le Brun N.E. J. Biol. Chem. 2003; 278: 17852-17858Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The function of ResB and ResC has not been studied on a molecular level but may lie in heme delivery and/or ligation to the apo-cytochrome. ResC contains a tryptophan-rich sequence motif, also found in system I CcmE and CcmF, thought to be involved in heme binding (18Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 19Le Brun N.E. Bengtsson J. Hederstedt L. Mol. Microbiol. 2000; 36: 638-650Crossref PubMed Scopus (79) Google Scholar, 40Goldman B.S. Beck D.L. Monika E.M. Kranz R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5003-5008Crossref PubMed Scopus (93) Google Scholar). ResA is a thiol-disulfide oxidoreductase with a very low midpoint redox potential (-340 mV at pH 7) that is tethered to the membrane via a single transmembrane helix (39Erlendsson L.S. Acheson R.M. Hederstedt L. Le Brun N.E. J. Biol. Chem. 2003; 278: 17852-17858Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). It is thought to reduce the disulfide bond of the Cys-Xxx-Xxx-Cys-His motif of the apo-cytochrome. The necessary electrons to do this are probably delivered by the integral membrane protein CcdA, a relative of DsbD (18Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 39Erlendsson L.S. Acheson R.M. Hederstedt L. Le Brun N.E. J. Biol. 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It also raises questions as to whether system II is indeed simpler than system I, whether each system II protein may combine functions of several system I proteins in one, and whether different pathways or underlying chemical principles may occur in the two CCM systems. To begin to address these issues, the three-dimensional structure of the enzymatic domain of ResA has been elucidated in its two biologically relevant redox states. These structures explain the molecular basis of the low redox potential that is fundamental to its function in reducing apo-cytochrome c. Moreover, the observed conformational differences in the two structures suggest a mechanism whereby the redox state of ResA might predetermine which substrate, apo-cytochrome c or CcdA, it binds. This selective substrate recognition may be a general feature of CCM-specific disulfide bond reduction systems.EXPERIMENTAL PROCEDURESSeleno-l-methionine Labeling of Soluble and His-tagged Soluble ResA—Histidine-tagged and wild-type soluble domain constructs of ResA were expressed in E. coli and purified as described previously (39Erlendsson L.S. Acheson R.M. Hederstedt L. Le Brun N.E. J. Biol. Chem. 2003; 278: 17852-17858Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). For over-expression and production of seleno-methionine-containing ResA, the E. coli selenium auxotroph strain B834(DE3) was transformed with plasmids pRAN8 (encoding His-tagged soluble ResA (htsResA)) or pRAN11 (encoding soluble ResA (sResA)) according to standard procedures (44Sambrook J. Fritsch F.E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The B834 strain cells were initially grown on M9 minimal media, supplemented with 50 μg/ml methionine, at 37 °C. When the cells reached an A600 = 1.0, the culture was centrifuged at 6000 × g for 10 min at 4 °C. Cells were resuspended in 1 liter of M9 minimal media without methionine, incubated at 37 °C, and centrifuged at 200 rpm. After 6 h, seleno-l-methionine was added to a final concentration of 50 μg/ml and the culture was incubated at 37 °C and centrifuged at 200 rpm for 30 min. Over-expression of ResA was then induced by adding isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 1 mm and the culture was incubated at 37 °C and centrifuged at 200 rpm for a further 10 h. Cells were harvested by centrifugation at 7000 × g for 15 min at 4 °C. The incorporation of seleno-methionine was verified by electro-spray mass spectrometry. Labeled htsResA was purified as described previously for the unlabelled proteins (39Erlendsson L.S. Acheson R.M. Hederstedt L. Le Brun N.E. J. Biol. Chem. 2003; 278: 17852-17858Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar).Crystallization, Data Collection, and Processing—As ResA was isolated as a mixture of 70% oxidized and 30% reduced states (39Erlendsson L.S. Acheson R.M. Hederstedt L. Le Brun N.E. J. Biol. Chem. 2003; 278: 17852-17858Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), complete oxidation of samples prior to crystallization was carried out by incubation in 10 mm diamide for 3 h at 277 K in the dark. Excess diamide was removed by using a 5-ml HiTrap desalting column that was previously equilibrated with 20 mm potassium phosphate, pH 7.0. Samples were subsequently concentrated to 12 mg/ml in 20 mm Na+/K+ phosphate, pH 7.0, and centrifuged for 10 min at 13,000 rpm at 277 K. Crystals were grown by the hanging-drop vapor diffusion method. Crystals of oxidized htsResA and sResA grew in approximately 2 weeks at 277 and 289 K from 24-27% (w/v) PEG 4000, 0.2 m ammonium acetate, and 0.1 m sodium citrate, pH 5.6-5.9. The optimal crystallization conditions for both protein forms were identical, and the resulting crystals were indistinguishable, although crystals of the His-tagged protein were not as reproducible. Two distinct crystal forms were obtained for both His-tagged and non-tagged proteins. The first type of crystals grew as extruded hexagonal needles that belonged to space group P65 with cell dimensions a = b = 36.6 Å, c = 176.9 Å with one molecule per asymmetric unit. The second crystal form also exhibited morphology strongly indicative of the underlying hexagonal symmetry. These bipyramidal hexagonal crystals belonged to space group P65 with cell dimensions a = b = 61.0 Å, c = 165.4 Å, with two monomers per asymmetric unit. Crystals of reduced ResA were obtained from similar conditions as the oxidized form with the addition of 10-40 mm dithiothreitol. These crystals grew in 2-3 days and belonged to space group P212121 with cell dimensions a = 47.5, b = 59.7, c = 110.1 Å. All ResA crystals could be frozen successfully in a solution containing 20% (v/v) ethylene glycol, 30% (w/v) PEG 4000, 0.2 m ammonium acetate, 0.1 m tri-sodium citrate, pH 5.6. The cryoprotectant solution of reduced crystals was supplemented with 40 mm dithiothreitol.Native data sets of the first, needle-like crystals of both htsResA and sResA were collected at 1.8 Å resolution at beam line XRD1 at Sincrotrone Trieste, Trieste, Italy. Data sets of the second type of oxidized ResA and of reduced ResA crystals were collected at 1.50 and 1.95 Å resolution, respectively, at BM14 of the European Synchrotron Radiation Facility, Grenoble, France. A multiple-wavelength anomalous dispersion (MAD) data set was collected at 2.37 Å resolution of seleno-methionine ResA crystals at the Protein Structure Factory, Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Berlin, Germany. All data sets were processed and reduced with the HKL package (45Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38349) Google Scholar). Data collection statistics are summarized in Table I.Table IMAD data for collection and refinement statistics for reduced and oxidized ResAOxidized Se-Met derivativeNative crystal dataPeakInflectionHigh energy remoteOxidized nativeReduced nativeData collectionBeam lineBESSY PSF BL1ESRF BM14ESRF BM14Wavelength (Å)0.97910.97930.91840.88560.8856Resolution (Å)30-2.3730-1.430-1.95Unique reflections27,75127,81327,82667,39823,398Space groupP65P65p212121Cell dimensions (Å)a = 61, b = 61, c = 165a = b = 61, c = 165a = 48, b = 60, c = 110Completeness (%) shellaValues in parentheses indicate the highest resolution shell.bValues in square brackets indicate the highest resolution shell used in refinement.99.5 (98.7)99.5 (97.8)99.7 (98.9)98.6 (86.4) [100]99.9 (99.1)Rsym (%)aValues in parentheses indicate the highest resolution shell.bValues in square brackets indicate the highest resolution shell used in refinement.7.8 (10.9)7.8 (10.4)6.2 (9.6)5.2 (44.6) [27.0]8.9 (27.3)I/σaValues in parentheses indicate the highest resolution shell.bValues in square brackets indicate the highest resolution shell used in refinement.13.2 (10.6)13.3 (10.8)12.7 (8.6)26 (2.0) [4.6]21.0 (5.3)Phasing methodMADMolecular replacementMolecular replacementFigure of meritcPhasing FOM before density modification.0.82Refinement and model statisticsResolution (Å)28.6-1.530-1.953Reflections used in Refinement55,50823,330Rfree, Rwork, Rcryst R (%)dR=|Fo−Fc|/Fo. Rfree is calculated with a 5% subset of the data that was not used for refinement; Rwork was calculated with the remaining 95% of the data. Rcryst, the crystallographic R factor, refers to the final model of ResA for which a final round of refinement was performed using all diffraction data.15.0, 12.3, 12.321.19, 18.31, 18.09Bond length r.m.s.d. (Å)eRoot-mean-square deviation from ideal stereo chemistry.0.0190.005Bond angle r.m.s.d. (°)eRoot-mean-square deviation from ideal stereo chemistry.1.681.34Protein atoms21992157Waters434300Other non-protein atoms160a Values in parentheses indicate the highest resolution shell.b Values in square brackets indicate the highest resolution shell used in refinement.c Phasing FOM before density modification.d R=|Fo−Fc|/Fo. Rfree is calculated with a 5% subset of the data that was not used for refinement; Rwork was calculated with the remaining 95% of the data. Rcryst, the crystallographic R factor, refers to the final model of ResA for which a final round of refinement was performed using all diffraction data.e Root-mean-square deviation from ideal stereo chemistry. Open table in a new tab Structure Determination and Refinement—The structure of untagged, oxidized ResA was determined by MAD using the anomalous signal of five incorporated selenium atoms per ResA molecule. Identification of the 10 Se atom substructure and subsequent phasing was carried out with SOLVE (46Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar). The obtained phases were of excellent quality (FOM = 0.82), which allowed for 87% of all amino acids in the asymmetric unit to be built automatically by RESOLVE (47Terwilliger T.C. Acta Crystallogr. Sect. D. 2003; 59: 38-44Crossref PubMed Scopus (593) Google Scholar, 48Terwilliger T.C. Acta Crystallogr. Sect. D. 2003; 59: 45-49Crossref PubMed Scopus (113) Google Scholar). The model was completed using alternating rounds of manual model building using O (49Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar) and automated refinement using programs of the CCP4 suite (50Collaborative Computational Project Number 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19702) Google Scholar, 51Potterton E. Briggs P. Turkenburg M. Dodson E. Acta. Crystallogr. Sect. D. 2003; 59: 1131-1137Crossref PubMed Scopus (1054) Google Scholar). As refinement of the protein model approached completion, refinement was continued using a 1.5 Å resolution native data set, and phases were calculated from the model. Water molecules were incorporated using ARP (52Lamzin V.S. Wilson K.S. Acta. Crystallogr. Sect. D. 1993; 49: 129-147Crossref PubMed Google Scholar) and, in the latter stages of refinement, the model was further improved by applying individual atomic anisotropic B-factor refinement using REFMAC (53Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta. Crystallogr. Sect. D. 1999; 55: 247-255Crossref PubMed Scopus (1005) Google Scholar).Throughout refinement, progress was monitored with the aid of an Rfree value calculated with 5% of the data (54Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3847) Google Scholar). Upon completion of the model (Rfree = 12.3%, Rwork = 15.0%), a final round of refinement using all of the data was used to give a single crystallographic R factor (Rcryst = 12.25%). The final model comprises 2 monomers of ResA (residues 37-173 and 39-173), 434 molecules of water, and 4 molecules of ethylene glycol. Several residues also exhibit clearly defined alternate conformations.The structure of reduced ResA was determined by molecular replacement using the CCP4 version of AMORE (55Navaza J. Acta Crystallogr. Sect. A. 1994; 43: 157-163Crossref Scopus (5027) Google Scholar) using as a search model the structure of the oxidized state in which the active-site cysteines were first replaced by alanines. A round of simulated annealing in CNS (56Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar) preceded iterative cycles of manual rebuilding using O (49Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar) and maximum likelihood refinement in CNS (57Adams P.D. Pannu N.S. Read R.J. Brunger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar). The addition of 300 waters and a final round of correlation-based refinement using all of the data completed the model (Rfree = 21.0%, Rwork = 18.3%, Rcryst = 18.1%). The reduced structure incorporates residues 39-175 in the first monomer and 39-174 in the second. Both the oxidized and reduced structures exhibit excellent stereochemistry and geometry as judged with PROCHECK (58Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Structure determination and refinement statistics are summarized in Table I.RESULTS AND DISCUSSIONHigh-resolution Structures of Oxidized and Reduced ResA—The crystal structure of oxidized ResA was determined at 2.37 Å by the MAD method using the anomalous signal of five incorporated selenium atoms per monomer and subsequently refined at 1.5 Å (Rcryst = 12.25%, Rwork = 12.3%, Rfree = 15.0%). The crystals contain two independent molecules in the asymmetric unit of a hexagonal P65 cell. These molecules can be superimposed with a root-mean-square deviation of 0.63 Å.The overall structure of monomeric ResA contains a classical thioredoxin fold, comprising a mixed four-stranded β-sheet surrounded by three helices. ResA contains two additions to this motif, one N-terminal β-hairpin (residues 36-63), and one insertion (residues 104-127), which gives rise to an additional strand and helix between strand β2 and helix α3 (Fig. 2A). Similar additions to the thioredoxin fold have been described recently in the structures of Bradyrhizobium japonicum CcmG (59Edeling M.A. Guddat L.W. Fabianek R.A. Thöny-Meyer L. Martin J.L. Structure. 2002; 10: 973-979Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and TlpA, which is essential for biosynthesis of the cytochrome aa3 oxidase from B. japonicum (60Capitani G. Rossmann R. Sargent D.F. Grutter M.G. Richmond T.J. Hennecke H. J. Mol. Biol. 2001; 311: 1037-1048Crossref PubMed Scopus (36) Google Scholar).Fig. 2The three-dimensional structure of a soluble and functional domain of ResA. A, three-dimensional structure of ResA. B, superposition of oxidized and reduced ResA. C, superposition of the two independent oxidized ResA molecules in the asymmetric unit. A, the protein exhibits a classical thioredoxin-like fold (blue) with two significant insertions: residues 37-63 contain a two-stranded, anti-parallel hairpin (green), whereas the central insert (residues 104-127, colored salmon) comprises one helix and one strand. Secondary structure elements are labeled from the N terminus, with the N-terminal transmembrane helix being α0. In the oxidized form, as displayed, a disulfide bond exists between the side chains of Cys-73 and Cys-76 (solid spheres). B, both oxidized and reduced structures are shown in a coiled representation. The oxidized state is shown in blue; the reduced form is shown in green. Positions of the Cys-73 Sγ and Cys-76 Sγ atoms in the oxidized state are shown as partially transparent yellow spheres. Several secondary structure elements are annotated. The most significant conformational differences between the two redox states are indicated by red circles. C, monomers are shown as yellow and blue coils, respectively. The second conformation of part of the insert observed in one monomer (yellow) is shown as a red coil. This and other protein structure figures were prepared with PyMOL (71DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar) and annotated with Paint Shop Pro 7 (Jasc).View Large Image Figure ViewerDownload (PPT)Crystals of dithiothreitol-reduced ResA belong to space group P212121 and contained two molecules per asymmetric unit. The structure of reduced ResA was determined by molecular replacement and refined at 1.95 Å resoluti
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