Structural Basis of Cellular Redox Regulation by Human TRP14

Abstract

Thioredoxin-related protein 14 (TRP14) is involved in regulating tumor necrosis factor-α-induced signaling pathways in a different manner from human thioredoxin 1 (Trx1). Here, we report the crystal structure of human TRP14 determined at 1.8-Å resolutions. The structure reveals a typical thioredoxin fold with characteristic structural features that account for the substrate specificity of the protein. The surface of TRP14 in the vicinity of the active site includes an extended loop and an additional α-helix, and the distribution of charged residues in the surface is different from Trx1. The distinctive dipeptide between the redox-active cysteines contributes to stabilizing the thiolate anion of the active site cysteine 43, increasing reactivity of the cysteine toward substrates. These structural differences in the active site suggest that TRP14 has evolved to regulate cellular redox signaling by recognizing a distinctive group of substrates that would complement the group of proteins regulated by Trx1. Thioredoxin-related protein 14 (TRP14) is involved in regulating tumor necrosis factor-α-induced signaling pathways in a different manner from human thioredoxin 1 (Trx1). Here, we report the crystal structure of human TRP14 determined at 1.8-Å resolutions. The structure reveals a typical thioredoxin fold with characteristic structural features that account for the substrate specificity of the protein. The surface of TRP14 in the vicinity of the active site includes an extended loop and an additional α-helix, and the distribution of charged residues in the surface is different from Trx1. The distinctive dipeptide between the redox-active cysteines contributes to stabilizing the thiolate anion of the active site cysteine 43, increasing reactivity of the cysteine toward substrates. These structural differences in the active site suggest that TRP14 has evolved to regulate cellular redox signaling by recognizing a distinctive group of substrates that would complement the group of proteins regulated by Trx1. Thioredoxin (Trx) 1The abbreviations used are: Trx, thioredoxin; TRP, thioredoxin-related protein; Prx, peroxiredoxin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MES, 4-morpholineethanesulfonic acid; Grx, glutaredoxin.1The abbreviations used are: Trx, thioredoxin; TRP, thioredoxin-related protein; Prx, peroxiredoxin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MES, 4-morpholineethanesulfonic acid; Grx, glutaredoxin. is a small redox protein that is ubiquitously distributed from Archaes to human (1Powis G. Montfort W.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 261-295Crossref PubMed Scopus (331) Google Scholar). In diverse organisms, it plays various physiological roles, acting as an electron donor and as a regulator of transcription and apoptosis as well as antioxidants (1Powis G. Montfort W.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 261-295Crossref PubMed Scopus (331) Google Scholar). Trx functions as a carrier that transfers electrons to enzymes involved in DNA synthesis and protein disulfide reduction: ribonucleotide reductase, methionine sulfoxide reductase (2Stubbe J. Riggs-Gelasco P. Trends Biochem. Sci. 1998; 23: 438-443Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 3Stadtman E.R. Moskovitz J. Berlett B.S. Levine R.L. Mol. Cell. Biochem. 2002; 234/235: 3-9Crossref Scopus (212) Google Scholar). Transcription factors such as NF-κB, p53, PEBP32, and AP-1 have been shown to be regulated by Trx (4Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (719) Google Scholar, 5Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (719) Google Scholar, 6Akamatsu Y. Ohno T. Hirota K. Kagoshima H. Yodoi J. Shigesada K. J. Biol. Chem. 1997; 272: 14497-14500Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Trx is also involved in the defense against oxidative stress through peroxiredoxins (Prxs) that directly remove cellular reactive oxygen species. Other functions of Trx include the regulation of apoptosis signaling kinase 1 (ASK1) to inhibit its activity (7Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2064) Google Scholar, 8Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1001) Google Scholar). Sequences of Trxs in various species are 27–69% identical to that of Escherichia coli, and all Trxs have the same three-dimensional fold, which consists of central five β-strands surrounded by four α-helices (9Eklund H. Gleason F.K. Holmgren A. Proteins. 1991; 11: 13-28Crossref PubMed Scopus (327) Google Scholar, 10Weichsel A. Gasdaska J.R. Powis G. Montfort W.R. Structure. 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The N-terminal cysteine of the Cys-Gly-Pro-Cys motif in Trx is redox-sensitive, and the motif is highly conserved. When compared with the general cysteine, the N-terminal cysteine has a lowered pKa value (11Holmgren A. Structure. 1995; 3: 239-243Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Various proteins sharing the Trx-like active site sequence have been found and classified as part of the Trx superfamily (12Matsuo Y. Hirota K. Nakamura H. Yodoi J. Drug News Perspect. 2002; 15: 575-580Crossref PubMed Scopus (11) Google Scholar). Among them, thioredoxin-related protein (TRP32) with the N-terminal thioredoxin domain has been found to bind to the catalytic fragment of mammalian STE-20-like kinase (13Lee K.K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-191664Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The active site sequence Cys-Gly-Pro-Cys of TRP32 is identical to that of thioredoxin. In contrast, nucleoredoxin (14Laughner B.J. Sehnke P.C. Ferl R.J. Plant Physiol. 1998; 118: 987-996Crossref PubMed Scopus (44) Google Scholar) and thioredoxin-related transmembrane protein (15Matsuo Y. Akiyama N. Nakamura H. Yodoi J. Noda M. Kizaka-Kondoh S. J. Biol. Chem. 2001; 276: 10032-10038Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), which seem to be involved in the various redox regulation, have modified sequences Cys-Pro-Pro-Cys and Cys-Pro-Ala-Cys, respectively. Recently, a novel thioredoxin-related protein (TRP14) was found from rat brain (16Jeong W. Yoon H.W. Lee S.R. Rhee S.G. J. Biol. Chem. 2004; 279: 3142-3150Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Jeong W. Chang T.S. Boja E.S. Fales H.M. Rhee S.G. J. Biol. Chem. 2004; 279: 3151-3159Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It is a 14-kDa cytosolic protein that contains a Cys-Pro-Asp-Cys motif with a sequence identity to human Trx (Trx1) of about 20%. TRP14 shows activity as disulfide reductase and takes up electrons from cytosolic thioredoxin reductase (TrxR1), like Trx1. However, TRP14 does not donate electrons to ribonucleotide reductase, methionine sulfoxide reductase, and Prxs, which are well known substrates of Trx1. TRP14 regulates TNF-α-induced signaling pathways in a different manner from Trx1. In RNA interference experiments, the depletion of TRP14 increased TNF-α-induced phosphorylation and degradation of IκBα more than the depletion of Trx1 did. TRP14 also facilitated activation of JNK and p38 MAP kinase induced by TNF-α. Unlike Trx1, TRP14 shows neither interaction nor interference with ASK1. Here, we determined the crystal structure of human TRP14 to understand the structural mechanisms for the substrate specificity of the protein and the biological role. The structure of TRP14 reveals a special surface topology with unique electrostatic surface properties near the active site that is quite different from Trx1. The distinctive surface properties of TRP14 explain the mechanism by which the target specificity is different between TRP14 and Trx1. In addition, the structural study of TRP14 provides us with information on the diversity and specificity of cellular redox regulation in the cytosol that are achieved by two distinctive Trx isotypes, TRP14 and Trx1. Protein Purification—The wild-type TRP14 protein was prepared as described (16Jeong W. Yoon H.W. Lee S.R. Rhee S.G. J. Biol. Chem. 2004; 279: 3142-3150Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, the harvested cells were dissolved in buffer A (20 mm Hepes (pH 7.0), 2 mm dithiothreitol, and 1 mm EDTA) and lysed by sonication. The pellet obtained by streptomycin sulfate and ammonium sulfate fractionation was dissolved in buffer A containing 0.5 m ammonium sulfate. Then, the protein fractions obtained by phenyl-Sepharose 6 FF were dialyzed against buffer B (10 mm Tris (pH 7.5), 2 mm dithiothreitol, and 1 mm EDTA) and applied to the Mono-Q column. The purified protein was dialyzed against 10 mm Hepes (pH 7.00) and concentrated to 20 mg/ml for crystallization. Crystallization and Data Collection—Crystallization of TRP14 was carried out by using the hanging drop vapor diffusion method at 18 °C. The reservoir solution consisted of 10%(w/v) polyethylene glycol 8000, 10%(w/v) polyethylene glycol monomethylether 2000, 0.1 m MES (pH 6.5). 1.8 μl of protein solution was mixed with an equal volume of the reservoir solution. Plate-shaped crystals grew at the full size of 1.0 × 0.5 × 0.2 mm in 1–2 days. The TRP14 crystals belonged to the P212121 space group with unit cell dimensions of a = 26.86 Å, b = 48.39 Å, c = 81.84 Å. The crystals contained one monomer in the asymmetric unit with the Matthews coefficient and the solvent content of 1.83 Å/Da and 34%, respectively. The multiwavelength anomalous dispersion data for the structure determination were collected in the beamline 18B at the Photon Factory by using an ADSC CCD detector. Before the data collection, a single crystal was soaked in stabilizing solution supplemented with 0.5 m sodium bromide for about 30 s and flash-frozen. Data for five different wavelengths including two peak wavelengths (λ1 and λ2), two edge wavelengths (λ1 and λ2), and a remote wavelength (λ5) were collected (Table I). Measurements for multiple peak and edge wavelengths were performed to take account of differences in the absorption edge of bromine between solution and crystalline states. All data were integrated using the program MOSFLM (18Leslie A.G.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1696-1702Crossref PubMed Scopus (485) Google Scholar) and scaled by using the program SCALA in CCP4 package (19Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19727) Google Scholar).Table ICrystallographic and refinement statisticsCrystallographic statisticsData setλ 1(peak)λ 2(peak)λ 3(edge)λ 4(edge)λ 5(remote)NativeWavelength(Å)0.91920.91950.91980.92010.91701.5416Resolution(Å)2.01.8Space group/unit cellP212121/26.79 X 47.95 X 79.99Rsym(%)aRsym = Σi |Ii - 〈I〉|/ Σ |〈I〉|, where I is the intensity for the Ith measurement of an equivalent reflection with the indices h, k, and l5.15.35.55.25.44.1I/σ (I)12.211.911.611.911.79.4Completeness(%)9999.7Total reflections64,16364,22064,02163,98364,49234,804(Unique reflections)7,3637,3747,3467,3517,38312,317Phasing powerbPhasing power = FH/E, where FH is the way heavy-atom structure factor amplitude and E is the lack-of-closure error(centric/acentric)-/-0.50/0.670.14/2.50.12/1.90.89/2.2(anomalous)2.22.32.51.92.2RculliscRcullis = (Σ |FPH ± FP| - FH) /Σ |FPH ± FH|, where FPH, FP, and FH are derivative, native and heavy-atom structure factor amplitudes, respectively(centric/acentric)-/-0.61/0.620.62/0.630.55/0.560.58/0.59(anomalous)0.800.790.760.860.81Refinement statisticsR-factor(%)dR-factor = Σ i |Fo - Fc|/Σ Fo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively21.9Rfree (%)eThe Rfree value was calculated from 5% of all data that were not used in the refinement22.9Bond lengths (Å)0.004Bond angles(°)1.224Dihedral angles(°)23.70Improper angles (°)0.75Ramachandran plotResidues in most favored regions (#/%)97/94.2Residues in additional allowed regions (#/%)5/4.8Residues in disallowed regions (#/%)1/1.0a Rsym = Σi |Ii - 〈I〉|/ Σ |〈I〉|, where I is the intensity for the Ith measurement of an equivalent reflection with the indices h, k, and lb Phasing power = FH/E, where FH is the way heavy-atom structure factor amplitude and E is the lack-of-closure errorc Rcullis = (Σ |FPH ± FP| - FH) /Σ |FPH ± FH|, where FPH, FP, and FH are derivative, native and heavy-atom structure factor amplitudes, respectivelyd R-factor = Σ i |Fo - Fc|/Σ Fo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectivelye The Rfree value was calculated from 5% of all data that were not used in the refinement Open table in a new tab Structure Solution, Model Building, and Refinement—The heavy atom search by using the program SnB (20Weeks C.M. Miller R. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 492-500Crossref PubMed Scopus (137) Google Scholar) located two bromide sites, and the heavy atom parameters were refined by using the program SHARP (21de La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The phases were further improved by solvent flattening by using the program (19Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19727) Google Scholar). A of and refinement is in The electron was of for the The program M. Acta Crystallogr. Sect. A. 1991; PubMed Scopus Google Scholar) was used for the When most residues in the were the the were to the data and refined by using the program P. J. M. R.J. T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; PubMed Scopus Google Scholar). The 5% of data was for Rfree were by the in In the refined of all residues in the are in the most favored and one in a disallowed of by the program R. A. M. and J. M. J. Crystallogr. Scholar). The has interaction with the in the and the of electron is The includes residues of TRP14 and of crystals were in 1 mm and mm (pH the crystals with the reservoir solution. The activity of TRP14 was by the of at a of to a containing mm (pH 7.0), 1 mm 0.2 mm and 6 The was performed at 30 and was on the of structure of TRP14 was determined by the multiwavelength anomalous dispersion method with a sodium bromide soaked TRP14 a and domain with the dimensions of × 30 × sequence identity between TRP14 and Trx, the structure of TRP14 is to that of When we TRP14 and Trx1 Data A. Gasdaska J.R. Powis G. Montfort W.R. Structure. 1996; 4: 735-751Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), out of be with a of The redox-active motif is of TRP14 is located at the A disulfide between the two cysteines was in the electron that the structure is in an sequence of TRP14 structural of human TRP14 and human Trx1 are and the respectively. with identity are and residues with a value in all TRP14 are as in the program Protein PubMed Scopus Google are structural differences between TRP14 and Trx1. the of TRP14 to of Trx1 is two and with the of residues and In addition, is toward the of the redox-active site A sequence shows that residues including and a part of in TRP14 be with Trx1. in TRP14 with the surface near the redox-active site a in target specificity by its interaction with Trx target proteins Other structural differences are found in loop that have an of residues in with Trx1 The extended loop of TRP14 is located in of the redox-active motif and is to a in target specificity as the and The extended loop to be by including between the oxygen of and the atom of and the between the oxygen atom of and the of In addition, the oxygen of with the of and active site motif in TRP14 is located at the of The N-terminal is to that the group of the be for the with substrates. The reactivity and redox of proteins are highly on the dipeptide sequence the The dipeptide sequence of TRP14 is unique in with that of Trx1 The of the dipeptide sequences between TRP14 and Trx1 to the pKa of the cysteine that is an of the reactivity of the redox-active The cysteine pKa of the proteins between and than that of the cysteine thiolate The lowered cysteine pKa is for the redox the of the cysteine reactivity toward substrates by increasing the of the In the thiolate anion is by the of the of as in Trx1. However, the of to be different between TRP14 and Trx1. In the unique dipeptide sequence between the two cysteines to to the pKa of the thiolate by the that is by of the two of the In Trx1, the dipeptide sequence is in which the of is different from that of The of Trx1 in the in the of a between the and the N-terminal cysteine In the in the of TRP14 does not the The of TRP14 in the does not the the of is not in the to a with the N-terminal cysteine The of the in is with the measurement of the pKa value of the N-terminal to be (16Jeong W. Yoon H.W. Lee S.R. Rhee S.G. J. Biol. Chem. 2004; 279: 3142-3150Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), which is than that of the cysteine of Trx The structure of an E. disulfide protein a J.L. Structure. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is known for an pKa value of The dipeptide sequence the motif of is to in in which the of is the same with and a the In the crystal structure of the thiolate in to in with of and the of plays a in the pKa value by a interaction with the of to residues such as and pKa from to that the in the plays a in the pKa of the cysteine in the motif R. M. T. J.L. Protein Sci. 1997; PubMed Scopus Google Scholar). surface the active site of TRP14 includes the and loop which is highly from the and surface of Trx1 and b and The and surface of Trx1 and would be for with a of substrates that to be by Trx1 and In the surface of TRP14 is to the interaction of proteins that not have a surface to the TRP14 In the between Trx1 and substrates that from J. J. Structure. 1996; 4: Full Text Full Text PDF PubMed Scopus Google Scholar) and J. J.R. Structure. 1995; 3: Full Text Full Text PDF PubMed Scopus Google Scholar), the target bind to the on the surface of the Trx1. In the the of in the to the site the active cysteine of the and the a N-terminal to the cysteine has with various residues and in Trx1. the is with Trx in a J. J. Structure. 1996; 4: Full Text Full Text PDF PubMed Scopus Google Scholar), the site is by in the and its are to of the However, in the the site and the of Trx substrates. In to the surface the distribution on the TRP14 active site surface is different from that of Trx1. the Trx1 surface is of that of TRP14 has highly charged with charged regions and and charged regions and Among the charged the of the motif plays an to a charged surface residues and are out in between the charged The highly charged surface of TRP14 as a for the of substrates. The surface of also has a small of charged residues the of the charged is than that of and the distribution of the charged residues is different between the two TRP14 to have evolved to a different of substrates that are not by Trx1 The structural and surface differences between TRP14 and cellular us to the TRP14 its biological the TRP14 crystals and the reductase activity of the protein by using as the substrate which with an was shown to be a substrate of the TRP14 (16Jeong W. Yoon H.W. Lee S.R. Rhee S.G. J. Biol. Chem. 2004; 279: 3142-3150Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In the TRP14 protein from crystals the same activity as the that the TRP14 observed in the crystal the active proteins as electron by electrons from to In the the proteins with electron and in a TRP14 electrons from Trx reductase 1 not from Trx reductase 2 Trx1 does from and (16Jeong W. Yoon H.W. Lee S.R. Rhee S.G. J. Biol. Chem. 2004; 279: 3142-3150Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The differences in specificity for the electron of TRP14 and Trx1 are more seem to be substrates between TRP14 and Trx1. Trx transfers electrons to ribonucleotide reductase, methionine sulfoxide reductase, and Prxs, TRP14 of the TRP14 was shown to with W. Chang T.S. Boja E.S. Fales H.M. Rhee S.G. J. Biol. Chem. 2004; 279: 3151-3159Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These distinctive substrate be for by the surface differences described in the is the of TRP14 to electrons from as does Trx1, the near the redox-active site of the two proteins are The reactivity toward be to the of with which would electrons from the active site to substrates Powis G. Biochem. J. PubMed Scopus Google Scholar). The of the would to TRP14 and Trx1 as substrates Powis G. Biochem. J. PubMed Scopus Google Scholar). also has the the cytosolic protein TRP14 not to have evolved to with which is a protein. were in which the substrate specificity of redox proteins was to differences in the surface The protein activity the active site surface of has like Trx1 M. G. Holmgren A. Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The crystal structure of that the specificity of electron was determined by the of J.R. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). sequences of human and H. are highly the active site are and the two proteins have different substrate in which human and H. Trx and as electron respectively. These that the active surface of redox proteins the substrate specificity of the proteins and the surface of TRP14 the protein with a highly in with its substrates. for is in cellular functions including and apoptosis in and Scholar). The by cellular redox are by of cysteine residues by reactive oxygen and the of the cysteines is by the known Trx1 has substrate it would be an for a protein to all of the proteins in mammalian cells have factors that complement the of Trx1. were known proteins and in where most cellular signaling TRP14 is a to an active as a redox regulator the protein electrons from TRP32 does not K. Nakamura H. H. Yodoi J. N. Y. Acad. Sci. 2002; PubMed Scopus Google Scholar). The structure of the cytosolic protein TRP14 reveals a charged and active site that is different from that of Trx1. However, the different active site surface be in the of two proteins as redox by the of substrates by the redox In to the surface TRP14 and Trx1 also differences in of the redox-active cysteines to a different dipeptide sequence between the two which the different redox properties of such as cysteine reactivity and redox the TRP14 structure shows that the protein is to as an factor for Trx1 and a redox regulator for cellular redox signaling N. and the of the Photon Factory beamline for with the data