Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m301526200
ISSN1083-351X
AutoresM.S. Alphey, Mads Gabrielsen, Elena Micossi, Gordon A. Leonard, Seán McSweeney, Raimond B. G. Ravelli, Emmanuel Tétaud, Alan H. Fairlamb, Charles S. Bond, William N. Hunter,
Tópico(s)Insect and Pesticide Research
ResumoTryparedoxin (TryX) is a member of the thioredoxin (TrX) fold family involved in the regulation of oxidative stress in parasitic trypanosomatids. Like TrX, TryX carries a characteristic Trp-Cys-Xaa-Xaa-Cys motif, which positions a redox-active disulfide underneath a tryptophan lid. We report the structure of a Crithidia fasciculata tryparedoxin isoform (CfTryX2) in two crystal forms and compare them with structures determined previously. Efforts to chemically generate crystals of reduced TryX1 were unsuccessful, and we carried out a novel experiment to break the redox-active disulfide, formed between Cys-40 and Cys-43, utilizing the intense x-radiation from a third generation synchrotron undulator beamline. A time course study of the S–S bond cleavage is reported with the structure of a TryX1 C43A mutant as the control. When freed from the constraints of a disulfide link to Cys-43, Cys-40 pivots to become slightly more solvent-accessible. In addition, we have determined the structure of Trypanosoma brucei TryX, which, influenced by the molecular packing in the crystal lattice, displays a significantly different orientation of the active site tryptophan lid. This structural change may be of functional significance when TryX interacts with tryparedoxin peroxidase, the final protein in the trypanothione-dependent peroxidase pathway. Comparisons with chloroplast TrX and its substrate fructose 1,6-bisphosphate phosphatase suggest that this movement may represent a general feature of redox regulation in the trypanothione and thioredoxin peroxidase pathways. Tryparedoxin (TryX) is a member of the thioredoxin (TrX) fold family involved in the regulation of oxidative stress in parasitic trypanosomatids. Like TrX, TryX carries a characteristic Trp-Cys-Xaa-Xaa-Cys motif, which positions a redox-active disulfide underneath a tryptophan lid. We report the structure of a Crithidia fasciculata tryparedoxin isoform (CfTryX2) in two crystal forms and compare them with structures determined previously. Efforts to chemically generate crystals of reduced TryX1 were unsuccessful, and we carried out a novel experiment to break the redox-active disulfide, formed between Cys-40 and Cys-43, utilizing the intense x-radiation from a third generation synchrotron undulator beamline. A time course study of the S–S bond cleavage is reported with the structure of a TryX1 C43A mutant as the control. When freed from the constraints of a disulfide link to Cys-43, Cys-40 pivots to become slightly more solvent-accessible. In addition, we have determined the structure of Trypanosoma brucei TryX, which, influenced by the molecular packing in the crystal lattice, displays a significantly different orientation of the active site tryptophan lid. This structural change may be of functional significance when TryX interacts with tryparedoxin peroxidase, the final protein in the trypanothione-dependent peroxidase pathway. Comparisons with chloroplast TrX and its substrate fructose 1,6-bisphosphate phosphatase suggest that this movement may represent a general feature of redox regulation in the trypanothione and thioredoxin peroxidase pathways. Tryparedoxin (TryX) 1The abbreviations used are: TryX, tryparedoxin; TryR, trypanothione reductase; TryP, tryparedoxin peroxidase; TrX, thioredoxin; MR, molecular replacement; ESRF, European Synchrotron Radiation Facility; FBPase, fructose-1,6-bisphosphatase; T[SH]2, the reduced form of trypanothione disulfide; Tb, T. brucei; Cf, C. fasciculata. is a thiol-disulfide oxidoreductase found in parasitic trypanosomatids belonging to the order Kinetoplastida. The principal biological function of TryX is to regulate oxidative stress as a component of the trypanothione peroxidase pathway (Fig. 1). This pathway, unique to trypanosomatids, starts with the NADPH-dependent trypanothione reductase (TryR) (1Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (695) Google Scholar), which maintains high levels of the polyamine-peptide conjugate trypanothione (N 1, N 8-bis(glutathionyl)spermidine) in the reduced form (T[SH]2), which in turn is able to reduce TryX (2Alphey M.S. Bond C.S. Tetaud E. Fairlamb A.H. Hunter W.N. J. Mol. Biol. 2000; 300: 903-916Crossref PubMed Scopus (143) Google Scholar, 3Hofmann B. Hecht H.-J. Flohé L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (778) Google Scholar). The reduced TryX interacts with and passes on a reducing equivalent to tryparedoxin peroxidase (TryP), allowing it to catalyze the reduction of hydrogen peroxide and organic hydroperoxides to water or alcohols, respectively. A feature of the trypanothione peroxidase pathway is that the shuttling of reducing power from TryR through to TryP utilizes the redox properties of disulfide linkages (4Raina S. Missiakas D. Annu. Rev. Microbiol. 1997; 51: 179-202Crossref PubMed Scopus (225) Google Scholar) in the enzymes and their peptide substrates. The first crystal structure of tryparedoxin, the protein from Crithidia fasciculata (CfTryX1), revealed a compact globular molecule classed in the same fold family as the functional homologue thioredoxin (TrX, see Fig. 2a) (5Alphey M.S. Leonard G.A. Gourley D.G. Tetaud E. Fairlamb A.H. Hunter W.N. J. Biol. Chem. 1999; 274: 25613-25622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The TrX fold is based on a twisted five-stranded central β-sheet with two helices on either side (6Katti S.K. LeMaster D.M. Eklund H. J. Mol. Biol. 1990; 212: 167-184Crossref PubMed Scopus (541) Google Scholar, 7Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (823) Google Scholar), but although classed in the same family, TryX is distinct from TrX in several respects (5Alphey M.S. Leonard G.A. Gourley D.G. Tetaud E. Fairlamb A.H. Hunter W.N. J. Biol. Chem. 1999; 274: 25613-25622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The parasite protein is significantly larger, ∼16 kDa as compared with 12 kDa for TrX, and carries additional elements of secondary structure, in particular a β-hairpin at the N terminus. The relationship of secondary structure with the amino acid sequence is so different for TryX and TrX that it is meaningless to compare the overall sequences. A noteworthy similarity is, however, the presence of a redox-active disulfide at the N terminus of an α-helix. In TrX, this disulfide is contained in the motif Trp-Cys-Gly/Ala-Pro-Cys, whereas in TryX, the motif is Trp-Cys-Pro-Pro-Cys. A least-squares fit of the central β-strands of TrX and TryX align these motifs on top of each other (5Alphey M.S. Leonard G.A. Gourley D.G. Tetaud E. Fairlamb A.H. Hunter W.N. J. Biol. Chem. 1999; 274: 25613-25622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In CfTryX1, the vicinal cysteine residues (Cys-40 and Cys-43) form a right-handed redox-active disulfide near the surface of the molecule, positioned beneath an overhanging Trp-39. Chemical modification and mass spectrometry studies indicate that the amino-proximal Cys-40 is the more reactive of the two cysteines (8Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohe L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar). Cys-40 is more solvent-accessible than the partner Cys-43 and actually forms a hydrogen bond with a water molecule (5Alphey M.S. Leonard G.A. Gourley D.G. Tetaud E. Fairlamb A.H. Hunter W.N. J. Biol. Chem. 1999; 274: 25613-25622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), the position of which suggests where T[SH]2 or TryP bind when reacting with CfTryX1. We now report structures for a second isoform of C. fasciculata TryX (CfTryX2) and provide brief comparisons with CfTryX1 and structures of the disulfide form and chemically reduced CfTryX2 recently published (9Hofmann B. Budde H. Bruns K. Guerrero S.A. Kalisz H.M. Menge U. Montemartini M. Nogoceke E. Steinert P. Wissing J.B. Flohé L. Hecht H.-J. Biol. Chem. 2001; 382: 459-471Crossref PubMed Scopus (54) Google Scholar). Unable to crystallize TryX as a homogeneous free dithiol form, we sought to generate this structure by photoreduction of the redox-active disulfide of CfTryX1 using the intense x-ray beam available from an undulator beamline at the European Synchrotron Radiation Facility (ESRF). As a control for this novel experiment and to study the effect of removing the disulfide linkage, Cys-43 was mutated to alanine, and the structure (CfTryX-C43A) was determined to 1.30-Å resolution. We also determined the structure of Trypanosoma brucei tryparedoxin (TbTryX) at 2.3-Å resolution, which presents an active site significantly different from any other TryX structure. Analysis of the interactions between TbTryX and a symmetry-related molecule suggests structural alterations that may be relevant to the interaction between TryX and the partner peroxidase, TryP. Based on crystal structures of a chloroplast TrX (10Capitani G. Markovic-Housley Z. DelVal G. Morris M. Jansonius J.N. Schurmann P. J. Mol. Biol. 2000; 302: 135-154Crossref PubMed Scopus (85) Google Scholar), one of its redox partners, fructose-1,6-bisphosphate phosphatase (11Chiadmi M. Navaza J. Miginiac-Maslow M. Jacquot J.-P. Cherfils J. EMBO J. 1999; 18: 6809-6815Crossref PubMed Scopus (109) Google Scholar), and our own sequence analyses, we propose that the conformational lability of the tryptophan lid may contribute to specific redox events. Cloning, Expression, and Purification of Recombinant Tryparedoxins—The gene coding for CfTryX2 was obtained by PCR amplification of genomic DNA of the C. fasciculata HS6 TryXII open reading frame (GenBank™ accession number: AF055986) using the oligonucleotides 5′-CAT CAT ATG TAT CAC ACC CTT CTC TAC-3′ for the sense strand and 5′-CAT GGA TCC TTA CTT CTT GGC CTC CAC GTT GGG-3′ for the antisense strand. The sense strand oligonucleotide contains an NdeI cloning site (underlined) incorporating an initiation codon (bold), whereas the antisense oligonucleotide contains a BamHI restriction site (underlined) downstream of the antisense stop codon (bold). The PCR products were blunt-end ligated into the SmaI site of pUC18 (Sure-Clone, Amersham Biosciences), and then the inserts were excised by restriction enzyme digest and ligated into the pET-15b vector (Novagen), creating plasmids pET-TbTryX and pET-CfTryXII. The mutagenesis of cysteine to alanine at residue 43 in C. fasciculata tryparedoxin-I (CfTryX-C43A) was performed using the method described by Deng and Nickoloff (12Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1096) Google Scholar) with the Chameleon kit (Stratagene). The plasmid pET-CfTryX1 (13Alphey M.S. Tetaud E. Gourley D.G. Fairlamb A.H. Hunter W.N. J. Struct. Biol. 1999; 126: 76-79Crossref PubMed Scopus (6) Google Scholar) provided the matrix, and the oligonucleotide was 5′-TGG TGC CCG CCG GCC CGC GGC TTC ACG-3′. The gene coding for TbTryX was obtained by PCR amplification of genomic DNA of the T. brucei 427 TryX open reading frame (GenBank™ accession number: AJ006403) using the oligonucleotides 5′-TTG CAT ATG TCT GGC CTC GCC AAG TAT-3′ for the sense strand and 5′-CAT CAT ATG TCA GTT GGG CCA CGG AAA GTT GGC-3′ for the antisense strand. The sense strand oligonucleotide carried an NdeI cloning site (underlined) incorporating an initiation codon (bold), whereas the antisense strand oligonucleotide carried an NdeI restriction site (underlined) just downstream of the antisense stop codon (bold). The integrity of the cloned genes was confirmed by sequencing. All recombinant proteins were expressed in Escherichia coli strain BL21 (DE3). Expression and purification protocols followed those published by Alphey et al. (13Alphey M.S. Tetaud E. Gourley D.G. Fairlamb A.H. Hunter W.N. J. Struct. Biol. 1999; 126: 76-79Crossref PubMed Scopus (6) Google Scholar) and involved the use of metal ion affinity chromatography to exploit the presence of the N-terminal histidine tag, which was introduced by using the pET-15b vector and which was subsequently removed by cleavage with thrombin (Amersham Biosciences). Protein concentration was determined spectrophotometrically at 280 nm using a theoretical extinction coefficient of 38030 m–1 cm–1 (14Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5132) Google Scholar), and purity was evaluated using SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Crystallization, Data Collection, and Data Processing—Crystals were grown using the hanging drop vapor diffusion setup, and diffraction data were processed, reduced, and scaled using the HKL (15Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38777) Google Scholar) and CCP4 suite of programs (see Table I) (16Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). Crystals of CfTryX1 and CfTryX-C43A were obtained using the published conditions (13Alphey M.S. Tetaud E. Gourley D.G. Fairlamb A.H. Hunter W.N. J. Struct. Biol. 1999; 126: 76-79Crossref PubMed Scopus (6) Google Scholar). Two tetragonal crystal forms (A and B) of CfTryX2 were obtained. Form A presented as rods and appeared in drops containing ∼10 mg ml–1 protein, 15% w/v polyethylene glycol 8000, 30 mm sodium cacodylate, pH 6.5, 5 mm dithiothreitol, and 60 mm ammonium sulfate. These crystals display space group P42212 with unit cell dimensions of a = b = 111.7, c = 56.5 Å and are isomorphous to samples studied by Hofmann et al. (9Hofmann B. Budde H. Bruns K. Guerrero S.A. Kalisz H.M. Menge U. Montemartini M. Nogoceke E. Steinert P. Wissing J.B. Flohé L. Hecht H.-J. Biol. Chem. 2001; 382: 459-471Crossref PubMed Scopus (54) Google Scholar). Form B crystals displayed a bipyramidal morphology and grew from solutions of ∼10 mg ml–1 protein, 500 mm sodium citrate, 30 mm sodium HEPES, pH 7.5, 5 mm dithiothreitol. They are in space group P41212 with unit cell dimensions of a = b = 114.3, c = 102.0 Å. Single crystals of both forms were cryo-protected by soaking in crystallization mother liquor containing either 15% (form A) or 10% (form B) of glycerol prior to transfer in a nitrogen gas stream at –170 °C. A single crystal of form A was used on the ESRF bending magnet beamline BM14, and data collection was carried out at λ = 0.977 Å to d min = 1.5 Å with an MarCCD133 detector in a single sweep totaling 90o of oscillation in 0.5o steps. For form B, a single crystal was mounted on the ESRF undulator beamline ID14-EH2, and data were collected at λ = 0.933 Å using an ADSC QUANTUM4 detector. Despite the relatively large size of the crystal used for data collection (∼250 × 150 × 150 μm3), diffraction maxima were only visible to ∼2.2-Å resolution, and these were only apparent after a relatively long exposure time of 45 s/0.5o oscillation. Radiation damage was evident after 75 images, and the crystal was translated such that a fresh section was exposed to the x-ray beam and a further 44 0.5o images were collected. Both batches of data were processed and scaled together yielding a data set complete to 2.35 Å.Table IData collection, refinement, and model geometry statistics Values in parentheses refer to the highest resolution bin.StructureTbTryXCfTryX2ACfTryX2BCfTryX-ACfTryX-BCfTryX-CCfTryX-C43AUpper resolution limit (Å)2.31.502.351.501.501.701.30No. of measurements/unique37965/4699469429/57687136117/28025167903/22718167593/22753116000/15786261452/35022Redundancy/completeness (%)8.1/98.7 (94.7)8.1/99.7 (95.5)4.9/97.5 (99.9)7.4/90.0 (46.7)7.4/94.5 (76.3)7.4/96.2 (93.6)7.5/96.6 (88.9)I/σ(I)19.6 (6.5)37.6 (2.1)29.8 (9.6)14.7 (1.2)16.8 (1.5)15.5 (1.4)18.6 (1.8)R merge (%)7.3 (21.7)4.5 (35.4)5.3 (24.9)5.2 (53.8)4.6 (39.5)6.1 (60.5)4.5 (43.9)Protein residues (total)144151aDenotes the two molecules per asymmetric unit./145aDenotes the two molecules per asymmetric unit.150aDenotes the two molecules per asymmetric unit./147aDenotes the two molecules per asymmetric unit.143143143143Water molecules/sulphates63/0312/113313312661210R-work/R-free (%)19.9/24.720.7/23.519.6/21.320.4/26.320.3/26.621.0/27.617.8/22.8Average isotropic thermal parameters (Å2)Wilson B-value37.519.052.217.418.222.214.4Overall20.623.148.721.422.526.520.3Main chain/side chains20.1/21.018.944.119.2/21.620.0/22.724.7/28.316.4/19.6Water molecules22.134.150.430.532.427.732.7r.m.s.d.bRoot mean square deviation. bond lengths (Å)/bond angles (°)0.007/1.450.022/1.860.021/1.650.014/1.50.013/1.40.016/1.70.012/1.6Ramachandran analysis (%)Favored regions88.387.186.894.393.491.892.6Additionally, generously allowed regions11.712.513.25.76.68.27.4Disallowed regions00.400000Protein Data Bank accession code1O731OC81OC91O7U1O851O8W1O8Xa Denotes the two molecules per asymmetric unit.b Root mean square deviation. Open table in a new tab For the time course experiment on CfTryX1 and the analysis of CfTryX-C43A, crystals were cryo-protected with 40% polyethylene glycol monomethyl ether 2000 and then flash-cooled at –170 °C. Data were collected on ID14-EH2 (λ = 0.933 Å) using the STRATEGY program (17Ravelli R.B. Sweet R.M. Skinner J.M. Duisenberg A.J.M. Kroon J. J. Appl. Crystallogr. 1997; 30: 551-554Crossref Scopus (61) Google Scholar) to determine the angular range for collection. For the time course experiment, a series of data sets were measured over the same oscillation range. Data sets A and B were consecutive and measured to a resolution of 1.5 Å. An intermediate exposure of 780 s, which corresponds to the total exposure time for measurement of a data set, was made while rotating the crystal, although no data were actually recorded. Data set C was then measured, and it was noted that the sample now only diffracted to 1.7-Å resolution. The radiation damage to the crystal after data set CfTryX-C was judged too great to warrant further useful data collection. The three data sets and the models derived from each are labeled CfTryX-A, -B, and -C, respectively. Clumps of small monoclinic plate-like crystals of TbTryX grew over a period of weeks in drops made by mixing a solution of 10 mg ml–1 protein, 50 mm HEPES, pH 7.5, with the reservoir solution of 30% polyethylene glycol 4000, 100 mm sodium acetate, pH 4.6, 200 mm ammonium acetate. The crystals display space group P21 with unit cell dimensions of a = 30.6, b = 31.5, c = 56.9 Å, b = 93.4°. The asymmetric unit comprises a monomer with ∼30% solvent content and V m of 1.8 Å3 Da–1. A small fragment (∼200 × 50 × 10 μm3) was removed from a clump of crystals and passed through a cryo-protectant consisting of reservoir solution adjusted to include 20% 2-methyl-2,4-pentanediol, and then transferred into a stream of nitrogen gas at –170 °C. Data were measured to 2.3-Å resolution on a Rigaku rotating anode (copper Kα λ = 1.5418 Å)-Raxis IV image plate system. Structure Solution and Refinement—The initial phases for both CfTryX2 structures were obtained using the molecular replacement (MR) technique as implemented in the CNS software package (18Brünger 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. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar) with data in the resolution ranges 15–3 Å for form A and 20–4 Å for form B. The structure of CfTryX1 (Protein Data Bank code 1QK8) (5Alphey M.S. Leonard G.A. Gourley D.G. Tetaud E. Fairlamb A.H. Hunter W.N. J. Biol. Chem. 1999; 274: 25613-25622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) stripped of all solvent molecules was used as the search model. After this procedure, it was clear that both crystal forms contain two molecules/asymmetric unit, which results in calculated Matthews coefficients (V m) (19Matthews B.M. J. Mol. Biol. 1976; 33: 491-497Crossref Scopus (8006) Google Scholar) of 2.3 and 4.4 Å3 Da–1 for forms A and B, respectively. The unit cell of form A has a much lower bulk solvent volume, 46%, than the 72% observed for form B, and this helps to explain the different diffraction limits of the two forms. For crystal form A, the initial MR phases obtained were extended to the resolution limit of the data set, 1.5 Å, using a combination of non-crystallographic symmetry averaging, solvent flattening, and histogram matching as implemented in the program DM (20Cowtan K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, 31. SERC Daresbury Laboratory, Warrington, United Kingdom1994: 34-38Google Scholar) after first calculating reliable σA-weighted figures-of-merit (FOM) (21Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2053) Google Scholar) for the MR phase set. The resulting electron density map (Fobs, αDM, FOMDM) was of excellent quality, and a model was constructed using the program ARP/wARP (22Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (484) Google Scholar). Refinement was then carried out using CNS interspersed with rounds of rebuilding in QUANTA (Accelrys) during which solvent molecules were included. To complete the refinement, a final round was performed using the program REFMAC (23Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (14025) Google Scholar) in which the two sulfur atoms in the active site were refined with anisotropic temperature factors. For crystal form B, a similar protocol to that described for form A was used to extend the MR phases to the diffraction limit of the data set. A model was built manually with QUANTA, and refinement carried out in a similar manner to that for form A. The CfTryX-A, -B, and -C structures are isomorphous with the disulfide form of CfTryX1 (5Alphey M.S. Leonard G.A. Gourley D.G. Tetaud E. Fairlamb A.H. Hunter W.N. J. Biol. Chem. 1999; 274: 25613-25622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), which provided the starting model for refinements using REFMAC. Following rigid body refinement, additional rounds of positional and B-factor refinement combined with graphics fitting (O) (24Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13038) Google Scholar) were carried out. Water molecules were added using ARP/wARP. Once the R-factor and R-free had dropped from about 40 to 25%, anisotropic B-factor refinement was introduced. The structure of TbTryX was solved by MR (AMoRe) (25Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5038) Google Scholar) using a poly-Ala structure of CfTryX1 as the search model. A clear solution was obtained that, after rigid body refinement, gave an R-factor of 48% and a correlation coefficient of 0.56 for data in the range of 30–2.3-Å resolution. Density modification (DM) improved the electron density map that was then used for model building. Simulated annealing molecular dynamics (to reduce model bias), least-squares refinement with CNS, together with the placement of water molecules completed the analysis. Approximately 5% of each data set was set aside to provide an R-free to monitor the progress of all refinements (26Brünger A.T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 24-36Crossref PubMed Google Scholar), whereas PROCHECK (27Laskowski R.A. MacArthur R.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and OOPS (28Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 829-832Crossref PubMed Scopus (164) Google Scholar) were used to assess model geometry. Further experimental details are provided (see Table I) and in the Protein Data Bank depositions. Overall Structures—The tryparedoxin structure is constructed around a seven-stranded twisted β-sheet with parallel and antiparallel alignments. This sheet starts with a β-hairpin formed by β1 and β2, and thereafter a β3-α1-β4-α2-β5 combination. A final β-hairpin between β6 and β7 completes the structure. The active site Trp-Cys-Pro-Pro-Cys motif is located between strand β3 and the N terminus of helix α1 (Fig. 2a). A structure-based sequence alignment of the three highly conserved tryparedoxins used in this study is shown in Fig. 2b. The Second Tryparedoxin Isoform of C. fasciculata (CfTryX2)—The structure of CfTryX2 was determined independently in two crystal forms, each of which presents two molecules/asymmetric unit. A pairwise least-squares superposition of all Cα atoms for the four molecules gave root mean square deviation values that ranged from 0.2 to 0.5 Å, and the results are similar whether we used a MR protocol or the anomalous dispersion from sulfur atoms (29Micossi E. Hunter W.N. Leonard G.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 21-28Crossref PubMed Scopus (60) Google Scholar) to provide the initial phase information. When comparing our MR-derived structures with those of CfTryX2 determined by Hofmann et al. (9Hofmann B. Budde H. Bruns K. Guerrero S.A. Kalisz H.M. Menge U. Montemartini M. Nogoceke E. Steinert P. Wissing J.B. Flohé L. Hecht H.-J. Biol. Chem. 2001; 382: 459-471Crossref PubMed Scopus (54) Google Scholar), least-squares superposition values of between 0.2 and 0.6 Å were observed. These values indicate close agreement of the second isoform structures irrespective of how or where they were determined or the redox state of the protein (see below). The structures reported here confirm that, when compared with the structure of CfTryX1, the helices α1 and α2 are closer to each other in the structure of CfTryX2, allowing the formation of a hydrogen bonding network around the less solvent-exposed sulfur atom in the active site S–S bridge (9Hofmann B. Budde H. Bruns K. Guerrero S.A. Kalisz H.M. Menge U. Montemartini M. Nogoceke E. Steinert P. Wissing J.B. Flohé L. Hecht H.-J. Biol. Chem. 2001; 382: 459-471Crossref PubMed Scopus (54) Google Scholar). Both crystal forms of CfTryX2 were grown from solutions containing dithiothreitol. The electron and difference density maps were suggestive of a time and space average of S–S bridge oxidized and reduced states. The refined S–S distances are 2.9 and 2.8 Å for the two molecules in form A and 3.2 and 3.0 Å for the two molecules in form B. Radiation-induced Cleavage of the Redox-active Disulfide— The exposure of protein crystals to an intense x-ray source changes the properties of the sample (30Burmeister W.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 328-341Crossref PubMed Scopus (365) Google Scholar, 31Ravelli R.B. McSweeney S.M. Structure. 2000; 8: 315-318Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 32Weik M. Ravelli R.B. Kryger G. McSweeney S. Raves M.L. Harel M. Gros P. Silman I. Kroon J. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 623-628Crossref PubMed Scopus (471) Google Scholar, 33Weik M. Berges J. Raves M.L. Gros P. McSweeney S. Siolman I. Sussman J.L. Houee-Levin C. Ravelli R.B. J. Synchrotron. Rad. 2002; 9: 342-346Crossref PubMed Scopus (84) Google Scholar). These changes, which include an increase in unit cell volume, a decreased resolution to which diffraction data can be observed, increased mosaicity, and an increased Wilson B-factor, are indicative of general radiation damage. It has also been noted that although atomic B-factor values increase for successive data sets, the change is not equally distributed for all atoms but rather occurs at glutamate, aspartate, and cysteine residues. In the latter case, it appears that disulfide breakage contributes significantly to this increase in B-factors. In general, only one cysteine in a disulfide actually moves during bond breakage, whereas its partner remains well fixed (31Ravelli R.B. McSweeney S.M. Structure. 2000; 8: 315-318Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Weik et al. (32Weik M. Ravelli R.B. Kryger G. McSweeney S. Raves M.L. Harel M. Gros P. Silman I. Kroon J. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 623-628Crossref PubMed Scopus (471) Google Scholar), in a study of x-ray-induced damage to Torpedo californica acetyl-cholinesterase observed that active site residues are among the most radiation-sensitive of residues and suggested, in a similar fashion to disulfide bonds, that these groups constituted “weak links” in protein structures. Our study on CfTryX1 targeted a redox-active disulfide, which should constitute an even weaker link than a structural disulfide. This is indeed the case since the radiation dose required to break this S–S bond is much less than that reported for structural S–S links in lysozyme for example (31Ravelli R.B. McSweeney S.M. Structure. 2000; 8: 315-318Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). During the time course experiment, from CfTryX-A to CfTryX-C (Table I), we noted general symptoms of radiation damage to the sample; resolution decreased, the B factors for consecutive data sets increased, the unit cell volumes increased from 136,800 to 136,930 to 137,340 Å3, and the mean fractional isomorphous differences also increased from 0.08 (A and B) to 0.12 (A and C). In the disulfide form of CfTryX1, the Cys-40–Cys-43 Sγ–Sγ distance is 2.2 Å. In CfTryX-A, the distance between the two Sγ atoms has increased to 2.5 Å. This is most likely due to partial reduction or damage caused by the high x-ray intensity of the undulator beamline. In CfTryX-B, the Sγ-Sγ distance is 2.8 Å, and in CfTryX-C, it has increased to 3.0 Å (Fig. 3). Similar results were obtained in the structure of CfTryX2 in the presence of 2-mercaptoethanol where the Sγ-Sγ distances for the t
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