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• W2009013863 abstract "•Crystal structures of the N-terminal two Trx domains of ERp46 were solved•SAXS analysis revealed an opened V-shape formed by three Trx domains in ERp46•Independent actions of ERp46 Trx domains on folding substrates were indicated•Different roles of ERp46 and PDI in oxidative protein folding are suggested The mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER. The mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER. Oxidative protein folding, coupled with disulfide bond formation, is critical for the proper synthesis of most secretory and cell-surface proteins. While and soon after they are synthesized, such proteins acquire their native structures in the ER, which has multiple pathways for catalyzing formation of disulfide bonds (Bulleid and Ellgaard, 2011Bulleid N.J. Ellgaard L. Multiple ways to make disulfides.Trends Biochem. Sci. 2011; 36: 485-492Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, Sato and Inaba, 2012Sato Y. Inaba K. Disulfide bond formation network in the three biological kingdoms, bacteria, fungi and mammals.FEBS J. 2012; 279: 2262-2271Crossref PubMed Scopus (54) Google Scholar). In the mammalian ER, more than 20 protein disulfide isomerase (PDI) family proteins (Hatahet et al., 2009Hatahet F. Ruddock L.W. Ahn K. Benham A. Craik D. Ellgaard L. Ferrari D. Ventura S. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation.Antioxid. Redox Signal. 2009; 11: 2807-2850Crossref PubMed Scopus (457) Google Scholar) and more than five PDI family oxidation enzymes (Araki and Inaba, 2012Araki K. Inaba K. Structure, mechanism, and evolution of Ero1 family enzymes.Antioxid. Redox Signal. 2012; 16: 790-799Crossref PubMed Scopus (64) Google Scholar) constitute a diverse network that greatly promotes oxidative folding of a variety of multi-disulfide proteins, including cell-surface receptors, immunoglobulins, blood coagulation factors, and peptide hormones such as growth factors and insulin. To fully understand the mechanisms underlying ER quality control, detailed functional characterizations of each oxidative pathway are essential. Recent extensive studies by us and others have clarified that whereas ER oxidoreductin-1α (Ero1α) oxidizes the canonical PDI specifically and efficiently (Inaba et al., 2010Inaba K. Masui S. Iida H. Vavassori S. Sitia R. Suzuki M. Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI.EMBO J. 2010; 29: 3330-3343Crossref PubMed Scopus (87) Google Scholar, Masui et al., 2011Masui S. Vavassori S. Fagioli C. Sitia R. Inaba K. Molecular bases of cyclic and specific disulfide interchange between human ERO1alpha protein and protein-disulfide isomerase (PDI).J. Biol. Chem. 2011; 286: 16261-16271Crossref PubMed Scopus (54) Google Scholar, Wang et al., 2009Wang L. Li S.J. Sidhu A. Zhu L. Liang Y. Freedman R.B. Wang C.C. Reconstitution of human Ero1-Lalpha/protein-disulfide isomerase oxidative folding pathway in vitro. Position-dependent differences in role between the a and a’ domains of protein-disulfide isomerase.J. Biol. Chem. 2009; 284: 199-206Crossref PubMed Scopus (116) Google Scholar), Prx4, another ER-resident oxidative enzyme, exhibits significant reactivity toward a broad range of PDI family members (Sato et al., 2013Sato Y. Kojima R. Okumura M. Hagiwara M. Masui S. Megawa K. Saiki M. Horibe M. Suzuki M. Inaba K. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.Sci. Rep. 2013; 3: 2456PubMed Google Scholar, Tavender et al., 2010Tavender T.J. Springate J.J. Bulleid N.J. Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum.EMBO J. 2010; 29: 4185-4197Crossref PubMed Scopus (171) Google Scholar). Prx4 has a particular preference for two PDI family members, ERp46 (also known as EndoPDI) and P5, both in vitro and in cultured cells. Importantly, the Prx4-ERp46 and Prx4-P5 oxidative pathways are dedicated to rapid but promiscuous disulfide introduction, suggesting that ERp46 and P5 efficiently catalyze formation of disulfide bonds but lack the ability to introduce native disulfide bonds selectively (Sato et al., 2013Sato Y. Kojima R. Okumura M. Hagiwara M. Masui S. Megawa K. Saiki M. Horibe M. Suzuki M. Inaba K. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.Sci. Rep. 2013; 3: 2456PubMed Google Scholar). PDI is superior to ERp46 and P5 with respect to selective introduction of native disulfide bonds and can also function as an effective proofreader of non-native disulfide bonds. ERp46/P5 and PDI play distinct roles, acting cooperatively to accelerate proper oxidative protein folding through a process that involves rapid disulfide introduction by ERp46/P5 followed by efficient isomerization of non-native disulfide bonds by PDI (Sato et al., 2013Sato Y. Kojima R. Okumura M. Hagiwara M. Masui S. Megawa K. Saiki M. Horibe M. Suzuki M. Inaba K. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.Sci. Rep. 2013; 3: 2456PubMed Google Scholar). Based on the primary sequence of ERp46, this enzyme is predicted to contain three Trx-like domains (Trx1, Trx2, and Trx3), each with a CGHC redox-active site, connected by linker loops of 20 or more amino acid residues (Figure 1A; Figure S1A available online). Although information about the physiological functions of ERp46 is limited, the enzyme has been reported to serve as a stress-survival factor under hypoxia in endothelial cells; furthermore, loss of ERp46 results in reduced secretion of adrenomedullin, endothelin-1, and membrane-bound CD105, suggesting that it is involved in oxidative protein folding (Sullivan et al., 2003Sullivan D.C. Huminiecki L. Moore J.W. Boyle J.J. Poulsom R. Creamer D. Barker J. Bicknell R. EndoPDI, a novel protein-disulfide isomerase-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor.J. Biol. Chem. 2003; 278: 47079-47088Crossref PubMed Scopus (139) Google Scholar). Accordingly, ERp46 is preferentially expressed in plasma cells that synthesize and secrete thousands of immunoglobulin M (IgM) pentamers, which contain ∼100 disulfide bonds each, per second (Wrammert et al., 2004Wrammert J. Källberg E. Leanderson T. Identification of a novel thioredoxin-related protein, PC-TRP, which is preferentially expressed in plasma cells.Eur. J. Immunol. 2004; 34: 137-146Crossref PubMed Scopus (19) Google Scholar). Similarly, ERp46 is involved in insulin production in cultured β cells (Alberti et al., 2009Alberti A. Karamessinis P. Peroulis M. Kypreou K. Kavvadas P. Pagakis S. Politis P.K. Charonis A. ERp46 is reduced by high glucose and regulates insulin content in pancreatic beta-cells.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E812-E821Crossref PubMed Scopus (25) Google Scholar). Proteomic analyses using cysteine-trapping mutants demonstrated that ERp46 forms mixed disulfide complexes with Prx4 and Ero1α, suggesting the occurrence of the Prx4-ERp46 and Ero1α-ERp46 oxidative pathways in human cells (Jessop et al., 2009Jessop C.E. Watkins R.H. Simmons J.J. Tasab M. Bulleid N.J. Protein disulphide isomerase family members show distinct substrate specificity: P5 is targeted to BiP client proteins.J. Cell Sci. 2009; 122: 4287-4295Crossref PubMed Scopus (140) Google Scholar). Thus, determination of the structure, functions, and mechanisms of operation of ERp46 are of both fundamental and medical importance. In this study, we investigated the structure and catalytic mechanisms of ERp46. Using crystallographic analyses, we determined the high-resolution structures of Trx1 and Trx2, and in small-angle X-ray scattering (SAXS) analyses, we determined the overall shape of ERp46 in its reduced and oxidized states in solution. The results demonstrated that the protein exhibits an extended and presumably flexible domain arrangement, irrespective of redox state. These structural features are unique to ERp46, which has a radically different molecular architecture from that of other PDI family members. In agreement with this structural insight, functional analyses demonstrated that each Trx domain acts independently on folding substrates in the Prx4-driven oxidative pathway. The strong preference of Prx4 for ERp46 and the specific oxidation of PDI by Ero1α enabled the efficient recycling of hydrogen peroxide generated by the Ero1α-PDI combination for subsequent re-use in Prx4 catalysis of ERp46 oxidation, which resulted in significant acceleration of proper oxidative protein folding. It is thus conceivable that the mammalian ER has evolved cooperative oxidative pathways involving PDI family members with different structures and different functional roles to ensure the efficient production of large quantities of secretory and cell-surface proteins. We first sought to crystallize the first and second Trx domains of human ERp46. By molecular replacement using the deposited structure of ERp46 Trx3 (Funkner et al., 2013Funkner A. Parthier C. Schutkowski M. Zerweck J. Lilie H. Gyrych N. Fischer G. Stubbs M.T. Ferrari D.M. Peptide binding by catalytic domains of the protein disulfide isomerase-related protein ERp46.J. Mol. Biol. 2013; 425: 1340-1362Crossref PubMed Scopus (18) Google Scholar, Gulerez et al., 2012Gulerez I.E. Kozlov G. Rosenauer A. Gehring K. Structure of the third catalytic domain of the protein disulfide isomerase ERp46. Acta Crystallogr. F.Struct. Biol. & Crystalliz. Comm. 2012; 68: 378-381Crossref PubMed Scopus (13) Google Scholar; Protein Data Bank [PDB] ID: 3UVT) as a search model, we eventually determined their structures at resolutions of 2.5 Å for Trx1 and 0.95 Å for Trx2 (Table 1). The Trx1 crystal belongs to the P21 space group and contains nine molecules in the asymmetric unit, seven of which are in the oxidized form and two are in the reduced form. The Trx2 crystal belongs to the P212121 space group, and the asymmetric unit contains one molecule with an oxidized active site. Both structures exhibit a prototypical thioredoxin fold composed of a four-stranded β sheet core and four surrounding α helices (Figure 1B, left and middle). As shown in Figure 1C, the backbone atoms of Trx1, Trx2, and Trx3 are almost superimposable on each other, indicating that the main chain structures are extremely similar. However, the electrostatic potential around the peptide binding site differed substantially between the domains: in Trx1 and Trx2, positively charged residues are concentrated near the peptide binding site, whereas in Trx3, the corresponding region is almost neutral (Figure 1B). This finding may account for our recent observation that Trx1 and Trx2 of ERp46 are more efficient substrates for Prx4 than Trx3 (Sato et al., 2013Sato Y. Kojima R. Okumura M. Hagiwara M. Masui S. Megawa K. Saiki M. Horibe M. Suzuki M. Inaba K. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.Sci. Rep. 2013; 3: 2456PubMed Google Scholar; see also Discussion).Table 1Data Collection and Structure DeterminationERp46 Trx1 (Mixture of Oxidized and Reduced Form)ERp46 Trx2 (Oxidized Form)ERp46 Trx2-Prx4 C-Terminal Peptide ComplexData CollectionBeamlineBL44XU at SPring-8BL44XU at SPring-8BL44XU at SPring-8Space groupP21P212121P1Cell dimensions (Å)a = 50.4, b = 94.7, c = 142.0α = 90.0°, β = 90.85°, γ = 90.0°a = 32.6, b = 39.1, c = 87.2α = β= γ = 90.0°a = 35.1, b = 36.4, c = 40.6α = 81.17°, β = 87.47°, γ = 85.40°Wavelength (Å)0.900000.700000.90000Resolution range (Å)35.50–2.50 (2.64–2.50)12.57–0.95 (1.00–0.95)26.78–0.92 (0.94–0.92)No. of total observations159,099511,605364,423No. of unique reflections45,51470,957126,643Completeness (%)98.2 (94.7)99.9 (100.0)92.4 (83.0)I/σ(I)7.1 (2.4)10.9 (3.4)26.4 (2.0)Multiplicity3.5 (3.3)7.2 (7.1)2.9 (1.9)RmergeaRmerge = ΣΣj|<I(h)> − I(h)j| / ΣΣj|<I(h)>|, where <I(h)> is the mean intensity of symmetry-equivalent reflections.0.144 (0.549)0.095 (0.564)0.053 (0.336)RefinementResolution range (Å)34.773–2.50012.476–0.9526.785–0.92No. of reflections45,48770,878126,561RworkbRwork = Σ(IIFp(obs)I − IFp(calc)II)/ΣIFp(obs)I.0.18800.12220.1396RfreecRfree = R-factor for a selected subset (5%) of reflections that was not included in prior refinement calculations.0.23460.13480.1580RmsdBond length (Å)0.0030.0120.013Bond angle (°)0.6801.3701.500Ramachandran analysisMost favored (%)98.3699.3100.0Allowed (%)1.640.70.0Disallowed (%)0.00.00.0The number in parentheses represents statistics in the highest-resolution shell.a Rmerge = ΣΣj|<I(h)> − I(h)j| / ΣΣj|<I(h)>|, where <I(h)> is the mean intensity of symmetry-equivalent reflections.b Rwork = Σ(IIFp(obs)I − IFp(calc)II)/ΣIFp(obs)I.c Rfree = R-factor for a selected subset (5%) of reflections that was not included in prior refinement calculations. Open table in a new tab The number in parentheses represents statistics in the highest-resolution shell. We recently reported that the Prx4 C-terminal segment immediately after His244 is responsible for the functional interaction with the N-terminal Trx domain (a0) of P5 (Sato et al., 2013Sato Y. Kojima R. Okumura M. Hagiwara M. Masui S. Megawa K. Saiki M. Horibe M. Suzuki M. Inaba K. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.Sci. Rep. 2013; 3: 2456PubMed Google Scholar). In the crystal structure of the P5 a0-Prx4 C-terminal peptide complex, the Prx4 region Glu246-Gly251 interacts with the peptide-binding site of P5 a0 via a mixed disulfide bond between a resolving cysteine (Cys248) of Prx4 and an active-site cysteine (Cys57) of P5 a0 (Figure S2A). To further understand and characterize the mode of interaction between Prx4 and PDI family Trx domains, we carried out a crystal-structure analysis of ERp46 Trx1 and Trx2 in complex with the same Prx4 C-terminal peptide used for the structure of the P5 a0-Prx4 peptide complex. We succeeded in solving the crystal structure of the ERp46 Trx2-Prx4 peptide complex (Figure 2A). Although the resolution was as high as 0.92 Å, the electron density of the Prx4 peptide was visible only for a limited region between His244 and Ala250, including Cys248, which forms an intermolecular disulfide bond with Cys217 of ERp46 Trx2 (Figure 2B). As in the case of the P5 a0-Prx4 peptide complex, the Prx4 peptide interacts with a peptide-binding site of ERp46 Trx2 via hydrogen bonding between the guanidinium group of Arg281 (Arg118 in P5 a0) and the carbonyl group of the main chain of Glu246 (Prx4; Figure 2C). Prx4-catalyzed oxidation of ERp46 Trx2 was greatly slowed down upon the mutation of Arg281Ala (Figure S3), suggesting the important role of this hydrogen bonding in the functional interplay between Prx4 and PDI family members (see also Discussion). However, there are small but significant differences in the interface structures of the P5 a0-Prx4 peptide and ERp46 Trx2-Prx4 peptide complexes. Whereas in the former the electrostatic interaction occurred between Glu246 (Prx4) and Arg118 (P5 a0), in the latter, the side chain of Glu246 was oriented in the opposite direction, resulting in the disappearance of the corresponding interaction (Figure 2C). In this connection, the backbone atoms of the N-terminal half of the Prx4 peptide bound to ERp46 Trx2 deviated significantly from those of the peptide bound to P5 a0 (Figure S2B), and the electron density deriving from His244 and Gly245 completely disappeared in the P5 a0-Prx4 peptide complex (Figure S2A). Such different structural features of the two complexes may partly explain our recent observation that Prx4 has an ∼5-fold lower affinity for ERp46 than for P5 (Sato et al., 2013Sato Y. Kojima R. Okumura M. Hagiwara M. Masui S. Megawa K. Saiki M. Horibe M. Suzuki M. Inaba K. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.Sci. Rep. 2013; 3: 2456PubMed Google Scholar). Using the crystal structures of decameric Prx4 and the ERp46 Trx2-Prx4 peptide complex, we modeled the structure of the Prx4 decamer-ERp46 Trx2 complex in silico (Figure 2D). Notably, the backbone atoms of the Prx4 peptide bound to ERp46 Trx2 were highly superimposable onto the corresponding atoms in decameric Prx4 (Figure 2D, inset), indicating that no large conformational changes are induced upon formation of a mixed disulfide intermediate complex between ERp46 Trx2 and Prx4. In other words, the C-terminal region of oxidized Prx4 assumes a conformation readily susceptible to nucleophilic attack by Cys217 of ERp46 Trx2. Taken together, the structures of these complexes reveal the common mechanism underlying the recognition of Trx domains of PDI family members by Prx4 and suggest that slight but significant differences in the conformations of the bound Prx4 C-terminal segment may account for differences in the affinities of PDI family members for Prx4. To determine the domain arrangement and the overall shape of full-length ERp46 in solution, we carried out SAXS analysis. Figure 3A displays the SAXS profiles for the reduced and oxidized forms of ERp46 at infinite dilution (see Figures S4A and S4B for more details of the concentration dependence). The innermost portion of the Guinier plots (Figure 3A, inset) was linear without any upward curvatures at low Q2. The radius of gyration Rg and the normalized forward intensity, I(0)/c, were determined from the slope and intercept of the linear fits. The Rg values at infinite dilution were estimated to be 41.6 ± 0.4 Å and 41.7 ± 0.4 Å for the reduced and oxidized forms, respectively (Table 2). While the molecular mass of the ERp46 construct used for the SAXS analysis is 47 kDa based on its amino acid sequence (Figure S1B), those estimated from I(0) using BSA as the standard are 51 kDa for reduced form and 52 kDa for oxidized form. The deviations of the estimated molecular mass from the theoretical value (9% to ∼10%) are within the range of the accuracy expected from the interprotein calibration (Akiyama, 2010Akiyama S. Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering.J. Appl. Cryst. 2010; 43: 237-243Crossref Scopus (18) Google Scholar) and may originate from a slight difference (∼0.01 ml/g) in partial specific volume between ERp46 and BSA. Taken together, both reduced and oxidized forms of ERp46 exist predominantly as a monomer with similar overall structure in solution. This observation is consistent with the recent finding obtained by analytical ultracentrifugation, which revealed that ERp46 exists as a monomer (Funkner et al., 2013Funkner A. Parthier C. Schutkowski M. Zerweck J. Lilie H. Gyrych N. Fischer G. Stubbs M.T. Ferrari D.M. Peptide binding by catalytic domains of the protein disulfide isomerase-related protein ERp46.J. Mol. Biol. 2013; 425: 1340-1362Crossref PubMed Scopus (18) Google Scholar).Table 2SAXS Structural ParametersRg (Å)aGuinier analysis using the Q range from 0.00975 to Qmax < 1.0 / Rg.Rg (Å)bEstimates in real space upon P(r) determination.I(0) (au)aGuinier analysis using the Q range from 0.00975 to Qmax < 1.0 / Rg.I(0) (au)bEstimates in real space upon P(r) determination.Dmax (Å)cMaximum dimension estimated by using GNOM package.Vp (Å3)dPorod volume.MM from I(0) (kDa)eMM calculated by using the I(0) value for BSA as the standard.MM from Vp (kDa)fMM calculated according to an empirical relationship (MM = VP / 1.65; Petoukhvov et al., 2012).MM (kDa)gTheoretical MM calculated according to amino acid sequences.Reduced ERP4641.6 ± 0.441.8 ± 0.216.68 ± 0.0716.60 ± 0.04137 ± 59.4 × 10451.0 ± 0.25747Oxidized ERP4641.7 ± 0.442.0 ± 0.217.09 ± 0.0616.97 ± 0.04141 ± 61.0 × 10552.2 ± 0.26147BSA27.9 ± 0.1nd21.70 ± 0.02ndndndndnd66.4MM, molecular mass; nd, not determined.a Guinier analysis using the Q range from 0.00975 to Qmax < 1.0 / Rg.b Estimates in real space upon P(r) determination.c Maximum dimension estimated by using GNOM package.d Porod volume.e MM calculated by using the I(0) value for BSA as the standard.f MM calculated according to an empirical relationship (MM = VP / 1.65; Petoukhvov et al., 2012Petoukhov M.V. Franke D. Shkmuatov A.V. Tria G. Kikhney A.G. Gajda M. Gorba C. Mertens H.D.T. Konarev P.V. Svergun D.I. New developments in the ATSAS program package for small-angle scattering data analysis.J. Appl. Cryst. 2012; 45: 342-350Crossref Scopus (1221) Google Scholar).g Theoretical MM calculated according to amino acid sequences. Open table in a new tab MM, molecular mass; nd, not determined. To gain insights into the molecular shape of full-length ERp46, we calculated a pair distribution function, P(r), using the measured SAXS curves (Figure 3B). The regularization parameter was determined by indirect transform methods using perceptual criteria (Svergun, 1991Svergun D.I. Mathematical, ethods in smail-angle scateering data analysis.J. Appl. Cryst. 1991; 24: 485-492Crossref Scopus (189) Google Scholar, Svergun, 1992Svergun D.I. Determination of the regularrization parameter in indirect-transform methods using perceptual criteria.J. Appl. Cryst. 1992; 25: 495-503Crossref Scopus (2757) Google Scholar). Because P(r) denotes the distributions of linear distances (r) between every pair of atoms in a particle, the most frequent r value and the largest r value (Dmax) can be determined from the P(r). The Dmax values of ERp46 were thus estimated to be 137 ± 5 Å for the reduced form and 141 ± 6 Å for the oxidized form (Table 2), suggesting the marginal difference in overall structure upon oxidation of the redox-active sites (Figure 3C). To draw the overall structure of ERp46 from the SAXS data in an objective manner, we used both the quasi-monodispersed and ensemble-refined models. The representative models built with rigid-body refinement (ribbon models in Figure 3C) and shape reconstructions (smooth envelopes in Figure 3C) demonstrate that the three Trx domains of ERp46 lie in a plane whose dimensions are ∼110 Å × ∼50 Å for the reduced form and ∼115 Å × ∼50 Å for the oxidized form (Figure 3D). Three Trx domains were located apart from each other with very few physical contacts between them, forming an opened V-shape (Figure 3C). Loosely packed domain arrangements implied a flexible nature of ERp46. Therefore, we next tried to refine ensemble-optimized models (Bernadó et al., 2007Bernadó P. Mylonas E. Petoukhov M.V. Blackledge M. Svergun D.I. Structural characterization of flexible proteins using small-angle X-ray scattering.J. Am. Chem. Soc. 2007; 129: 5656-5664Crossref PubMed Scopus (846) Google Scholar) of reduced and oxidized forms of ERp46 using the crystallographic and SAXS data (Figure S5). However, the ensemble models gave rise to only a marginal improvement compared with the fitting using a single rigid-body or shape-reconstruction model (Table 3). Thus, given the flexible nature of ERp46, the degree of conformational freedom is likely limited, and the rigid body and shape reconstruction models can be treated as the representatives of accessible conformational states of ERp46 in solution.Table 3SAXS Shape Reconstruction Statistics for Reduced and Oxidized ERp46 without the N-Terminal Signal SequenceReduced ERp46Oxidized ERp46Shape reconstructionGASBOR2.2iGASBOR2.2iQ range (Å−1)0.01002–0.405200.01002–0.40520Real space range (Å)0–1360–139SymmetryP1P1Search spacespheresphereNo. of Shannon channels17.6717.67Total number of dummy residues421421SQRT(χ2) (mean ± SD)1.244–1.385 (1.294 ± 0.047)1.151–1.212 (1.182 ± 0.022)No. of models averaged1010DAMAVER NSD (mean ± SD)1.207–1.333 (1.271 ± 0.039)1.197–1.318 (1.236 ± 0.038)Shape reconstructionBUNCH08BUNCH08Q range (Å−1)0.00975–0.405500.00975–0.40550Real space range (Å)0–1380–148SymmetryP1P1Total number of residues421421Total number of dummy residuesaThe residues are numbered based on the amino acid sequence of recombinant ERp46 used for the SAXS measurement (Figure S1B).100100Dummy residuesaThe residues are numbered based on the amino acid sequence of recombinant ERp46 used for the SAXS measurement (Figure S1B).1–50, 160–178, 285–311, 418–4211–50, 160–178, 285–311, 418–421Total no. of known residuesaThe residues are numbered based on the amino acid sequence of recombinant ERp46 used for the SAXS measurement (Figure S1B).321321Known residues treated as rigid bodiesaThe residues are numbered based on the amino acid sequence of recombinant ERp46 used for the SAXS measurement (Figure S1B).51–159, 179–284, 312–41751–159, 179–284, 312–417SQRT(χ2) (mean ± SD)1.181–1.237 (1.216 ± 0.017)1.168–1.240 (1.190 ± 0.024)No. of models reconstructed1010DAMAVER NSD (mean ± SD)1.396–1.678 (1.478 ± 0.090)1.443–1.729 (1.574 ± 0.089)Ensemble reconstructionEOM2.0EOM2.0Q range (Å−1)0.00975–0.405500.00975–0.40550Real space range (Å)0–2240–242SymmetryP1P1No. of theoretical curves (pool structures)10,00010,000SQRT(χ2) (mean ± SD)1.157–1.159 (1.158 ± 0.001)1.107–1.110 (1.109 ± 0.001)Number of ensembles reconstructed55a The residues are numbered based on the amino acid sequence of recombinant ERp46 used for the SAXS measurement (Figure S1B). Open table in a new tab Such separated arrangements of Trx domains are characteristic of ERp46, which has unusually long interdomain linkers and a molecular architecture that is radically different from the known structures of other PDI family members. Notably, the redox-active CXXC motifs of ERp46 Trxs are solvent-exposed and separated from each other (Figure 3D). On the other hand, in PDI and ERp57, the redox-active sites are positioned so that they face each other across the central cleft of the overall “U”-like shape (Dong et al., 2009Dong G. Wearsch P.A. Peaper D.R. Cresswell P. Reinisch K.M. Insights into MHC class I peptide loading from the structure of the tapasin-ERp57 thiol oxidoreductase heterodimer.Immunity. 2009; 30: 21-32Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, Tian et al., 2006Tian G. Xiang S. Noiva R. Lennarz W.J. Schindelin H. The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites.Cell. 2006; 124: 61-73Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, Wang et al., 2012Wang C. Yu J. Huo L. Wang L. Feng W. Wang C.C. Human protein-disulfide isomerase is a redox-regulated chaperone activated by oxidation of domain a’.J. Biol. Chem. 2012; 287: 1139-1149Crossref PubMed Scopus (85) Google Scholar). The different distribution patterns of the redox-active sites in PDI family members lead us to speculate that these proteins may play different functional roles and interact with substrate proteins via different mechanisms (see also the next section). Using the representative rigid-body model of full-length ERp46 and a model structure of the ERp46 Trx2-Prx4 decamer complex (Figure 2D), we modeled three versions of the ERp46-Prx4 decamer complex, in which Trx1, Trx2, and Trx3 were each placed proximal to the C-terminal region of Prx4 so that they were superposed onto ERp46 Trx2 bound to the Prx4 decamer with the minimal root-mean-square deviation (rmsd; Figure S6). The modeled structures suggested that Trx3 makes slight contact with a part of Prx4 when Trx2 of full-length ERp46 was superimposed to Trx2 bound to Prx4 decamer (Figure S6A). Except for this, there are no gross steric hindrances between Prx4 and ERp46 in any version of the complex. Consistent with this, each Trx domain of ERp46 was oxidized at almost the same rate regardless of whether it was present on an isolated peptide or part of the whole protein (Sato et al., 2013Sato Y. Kojima R. Okumura M. Hagiwara M. Masui S. Megawa K. Saiki M. Horibe M. Suzuki M. Inaba K. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein foldi" @default.
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• W2009013863 date "2014-03-01" @default.
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• W2009013863 title "Radically Different Thioredoxin Domain Arrangement of ERp46, an Efficient Disulfide Bond Introducer of the Mammalian PDI Family" @default.
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• W2009013863 doi "https://doi.org/10.1016/j.str.2013.12.013" @default.
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