A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p

A screen for proteins that restore viability to an Ero1p mutant when overexpressed in yeast uncovered Erv2p⁶. Although Erv2p is normally expressed only under certain growth conditions⁷ and deletion of the ERV2 gene is not lethal⁷, Erv2p expressed under a heterologous promoter efficiently rescues the temperature- sensitive ero1-1 mutant at the nonpermissive temperature⁶. Prior to these experiments, Erv2p had been recognized^{7, 8} as a homolog of the essential for respiration and viability 1 protein (Erv1p), a mitochondrial FAD-binding sulfhydryl oxidase⁹. In recent years, additional homologs have been discovered, including a hepatotrophic growth factor called ALR (augmenter of liver regeneration)¹⁰ and virally encoded enzymes that promote disulfide-bond formation in poxvirus coat proteins¹¹, which assemble in the reducing environment of the cell cytosol. A domain homologous to Erv2p is fused to a thioredoxin-like domain in a family of proteins¹² that includes an egg white sulfhydryl oxidase¹³ and Quiescin Q6, a protein that is highly expressed¹⁴ and secreted¹⁵ when fibroblasts enter quiescence. Although enzymes bearing the Erv-like domain differ in their protein substrates, a key feature of these enzymes appears to be the ability to catalyze the reaction 2R-SH + O₂ R-S-S-R + H₂O₂. Thus, the basic sulfhydryl oxidase module of the Erv2p family can exist in a variety of compartmental and quaternary structural contexts to serve diverse physiological functions.

The Erv2p family has unusual features for sulfhydryl oxidoreductase flavoproteins. According to sequence alignments, the FAD-binding domain appears to comprise only 100 residues, making it considerably smaller than typical flavin binding oxidoreductases¹¹. Furthermore, the secondary structure prediction for Erv2p suggested a highly -helical protein¹¹. In contrast, classical FAD-binding folds¹⁶ and the thioredoxin fold contained within other known endoplasmic reticulum (ER)^{17, 18} and bacterial periplasmic^{19, 20} thiol oxidoreductases are mixed / structures. Finally, FAD-binding thiol oxidoreductases that have been studied in depth, such as thioredoxin reductase, glutathione reductase and dihydrolipoamide dehydrogenase, use NADH (or NAD⁺) as a substrate, whereas the Erv2p family uses O₂ as an electron acceptor. For the above reasons, Erv2p-like proteins differ from the flavoprotein families as currently described^{21, 22}.

To begin to elucidate the mechanism of the newly described class of disulfide bond donor enzymes to which Erv2p belongs and to further our understanding of the origin of the disulfide-rich environment of the ER, we determined the structure of Erv2p by X-ray crystallography. Erv2p retains its N-terminal signal sequence in vivo and remains fixed in the ER membrane⁶. For crystallization, we expressed a soluble fragment of Erv2p, called Erv2-N, which lacks the signal sequence and begins at Glu 30 (Fig. 1). The first crystals obtained were found to be proteolytically cleaved to a shorter fragment beginning in the vicinity of Thr 67, as determined by N-terminal peptide sequencing of the major degradation product in the crystallization stock solution. Data were collected to 2.0 Å resolution from these crystals, which were of space group P2₁ with unit cell dimensions a = 53.6 Å, b = 66.8 Å, c = 60.4 Å and = 91.4°, and four molecules per asymmetric unit.

Figure 1. Sequence and secondary structure of Erv2p. The section of Erv2p corresponding to the structure determined by crystallography is shown in white lettering on a black background. Residues not present in the structure are in black on a white background. Black dots highlight every 10th residue. Helices are indicated by rods under the sequence, and disulfide bonds are shown by dotted lines between Cys residues. Bent arrows indicate the N-terminus of the expression construct Erv2-N, which extends to the natural C-terminus of the protein, and the limits of the thermolysin-cleaved fragment Erv2-c. Different preparations of Erv2-c had either Leu 71 or Met 72 as the predominant N-terminal residue. Full Figure and legend (32K)

To increase the reproducibility of crystallization, a limited proteolysis step was added to the Erv2-N purification procedure. Cleavage with thermolysin removed an N-terminal fragment, leaving a protease-resistant domain. The predominant N-terminal residue of this stable product was Leu 71, and the molecular weight obtained by mass spectrometry was 13,395 10 Da. This mass is consistent with Ser 187 being the C-terminal residue of the protease-resistant domain (Fig. 1), which gives a calculated mass of 13,390 Da. This fragment spans the region of high sequence homology with other proteins of the family and will be referred to as Erv2-core (Erv2-c).

The protease-resistant fragment Erv2-c was compared by circular dichroism spectroscopy (CD) to Erv2-N. Both intact and truncated enzymes gave CD spectra typical of highly helical proteins (Fig. 2). Subtraction of the spectrum for Erv2-c from that of Erv2-N yielded a difference spectrum typical of an irregular structure. The lack of fixed secondary structure and the extreme protease sensitivity of the N-terminal 40 residues of Erv2-N suggest that this region is flexible. Thus, Erv2-c comprises the structural core of the Erv2p protein. The FAD bound to Erv2-c is sensitive to reduction by concentrations of dithiothreitol (DTT) that do not reduce free flavin, demonstrating that Erv2-c is catalytically active (data not shown).

Figure 2. CD of Erv2-N and Erv2-c. The CD signal as a function of wavelength is displayed for Erv2p lacking only the signal sequence (Erv2-N; filled circles) and for thermolysin-cleaved enzyme (Erv2-c; open circles) at the same concentration. Both spectra are typical for highly helical proteins. The difference between these two spectra (inset) is indicative of random coil, demonstrating that the regions of Erv2p absent from the crystallographic study lack regular secondary structure. Full Figure and legend (16K)

Erv2-c crystals nucleated poorly but grew rapidly under a variety of conditions when seeded from existing crystals. Data were collected to 1.5 Å from a crystal of space group P2₁, unit cell dimensions a = 47.6 Å, b = 45.2 Å, c = 53.8 Å and = 100.2°, with two molecules per asymmetric unit. Crystals were grown with SeMet in place of the single methionine residue in the domain, and phases were obtained by multiwavelength anomalous dispersion (MAD). The structure built into the MAD electron density maps of Erv2-c was used in molecular replacement to provide phases for diffraction data from the crystal of degraded Erv2-N. The structures were then refined independently for each crystal form.

In total, six copies of the Erv2p protomer were observed between the two crystal forms, distributed as two dimers in the crystals grown from the Erv2-N preparation (one is shown in Fig. 3) and one dimer in the crystals of Erv2-c. Most of Erv2-c is structurally similar in all six protomers; the region between Asp 75 and Tyr 174 has an average root mean square (r.m.s.) deviation of 0.30 Å for backbone atoms. This region contains a four-helix bundle (helices 1−4) and an additional single turn of helix (5) packed perpendicular to the bundle (Fig. 3c). The 4 helix (Arg 142−Tyr 159) crosses 3 at an angle of 50°, which is typical of helix-crossing angles in globular proteins but not in four-helix bundles²³. The 3 helix (Gly 122−Lys 134) is at an 30° angle to 2, and 4 makes an 20° angle with 1. The C-terminus of 4 splays out from the N-terminus of 1, and FAD binds between these helices (Fig. 3c). The 1 and 2 helices (Asp 75−Ala 94 and Pro 102−Leu 118, respectively) are nearly antiparallel to one another and form the dimer interface by packing against the symmetry-related helices at an angle of 55°. The dimer interface is hydrophobic with a contact area of 700 Ų, and the hydrophobic nature of 10 residues in the interface is conserved among homologs.

Figure 3. Structure of Erv2p. a, Stereo diagram of an Erv2-N dimer viewed down the approximate two-fold symmetry axis. Every 10th residue in one protomer is indicated by a dot and labeled according to its position in the full Erv2p sequence. This figure was generated using MOLSCRIPT47. b, A ribbon diagram of the dimer in the same orientation as the stereo diagram shows one protomer in red and the second in blue. Cys side chains are illustrated in ball-and-stick representation and numbered. The two FAD molecules bound by the dimer are shown as balls and sticks. The N- and C-termini of each chain is labeled 'N' and 'C,' respectively. This and the following structure figures were generated with RIBBONS48. c, View down the four-helix bundle of one subunit, with helices labeled 1−5 according to their positions from the N-terminus to the C-terminus along the polypeptide chain. Full Figure and legend (68K)

A search using DALI²⁴ for folds similar to that of Erv2p returned relatively weak matches with other four-helix bundle proteins. The three highest-scoring proteins were the transmembrane subunit c of ATP synthase (PDB accession code 1C17, Z-score 4.5, r.m.s. deviation 2.9 Å over 76 residues), cytochrome c' (1CPQ, Z-score 4.1, r.m.s. deviation 4.1 Å over 81 residues) and myohemerythrin (2MHR, Z-score 3.9, r.m.s. deviation 3.4 Å over 71 residues). The first match is likely to arise from the fortuitous convergence of topology and helix−helix crossing angles in two proteins that are otherwise unrelated (ATP synthase subunit c helices are much longer and are membrane-imbedded). Cytochrome c' and myohemerythrin are more similar to Erv2-c in that they are soluble and bind cofactors. Furthermore, cytochrome c' is involved in electron transport and myohemerythrin transports oxygen, whereas Erv2p transfers electrons from sulfhydryl groups to oxygen. However, Erv2p helix 3 aligns very poorly to cytochrome c' and myohemerythrin, and Erv2p appears to be an atypical member of the four-helix bundle fold family. To our knowledge, Erv2p is the first FAD-binding protein found to belong to the 'all ' fold class.

Figure 4. FAD binding site. a, A ribbon diagram of the polypeptide backbone with some side chains and the bound FAD shown in ball-and-stick representation. The bent conformation of the FAD buries the isoalloxazine ring and adenine portions of the cofactor while keeping the intervening regions surface exposed. b, The ribbon trace of the polypeptide backbone has been removed in this panel so that the residues involved in aromatic ring stacking with the FAD can be identified. Full Figure and legend (57K)

Of the six Cys residues in Erv2p, only the two in the active-site Cys-X-X-Cys motif are conserved in every known family member. The Erv2p Cys-Gly-Glu-Cys sequence, like the Cys-X-X-Cys motif in thioredoxin, is found at the N-terminus of a helix — in this case, 3 — and these Cys residues are disulfide-bonded to each other (Fig. 5). Similar to Escherichia coli thioredoxin reductase²⁵, but different from glutathione reductase²⁶, the Erv2p active site Cys residues are on the re face of the FAD isoalloxazine ring (Fig. 5). Erv1p, Erv2p and ALR, but not quiescin or the viral enzymes, share a second Cys pair found at positions 150 and 167 in Erv2p. These Cys residues form a disulfide bond near the adenine portion of the FAD and fix the short C-terminal helix 5 against the side of helix 4 (Fig. 3b,c).

Figure 5. Electron density map in the vicinity of the Erv2p active site. A combined simulated annealing omit map calculated with CNS45 is displayed at 1.2 around the final model for the Erv2-c crystals. The Tyr-Pro-Cys-X-X-Cys motif is conserved among Erv1p, Erv2p and ALR. In contrast, the residues corresponding to the Xs are highly variable, and these positions are surface exposed. Electron density for the side chain of Glu 123 is almost entirely lacking, indicating that this amino acid is flexible. Full Figure and legend (63K)

Although residues after Ser 179 in the Erv2-c structure could not be traced in the electron density maps, the region from Asp 175 to Ser 179, including the disulfide bond between Cys 176 and Cys 178, was clearly observed in either of two conformations (Fig. 6). In three of the six copies of the protomer (both chains in one dimer and one chain of another dimer), this C-terminal tail swings away from the dimer interface, and the Cys-Gly-Cys region packs back against the 5 helix of the same protomer. In the remaining three molecules, the tail extends away from the 5 helix and lies across the active site of the opposite subunit in the dimer. The C atom of Cys 178 approaches within 4 Å of the C of Cys 121 (as opposed to 14 Å in the alternate conformation), and a mere change in the side chain torsion angles of these residues would permit formation of a disulfide bond between them (Fig. 6, inset).

Figure 6. Erv2p flexible C-terminal tail. The four molecules in the asymmetric unit of the crystals grown from the original Erv2-N preparation were superposed to compare the orientations of the C-terminal regions containing the Cys-Gly-Cys sequence. In this crystal, one of the four C-terminal tails packs against the neighboring active site. The arrow indicates the significant conformational differences between the superposed molecules. Shown in the inset is a model of an intersubunit disulfide bond constructed by changing the 1 side chain torsion angles of Cys 178 and Cys 121 to decrease the sulfur−sulfur bond distance between these residues to 2.03 Å. Full Figure and legend (59K)

Figure 7. Cys residues required for disulfide bond formation between Erv2p protomers and for Erv2p function in vivo. Cells overproducing wild type or Ala-substitution mutants of Erv2p-HA were labeled with [35S]methionine and then lysed in 10% (w/v) TCA to block disulfide exchange. Free thiols were alkylated with NEM prior to immunoprecipitation with HA antibody. a, Wild type Erv2p exists in monomeric and dimeric forms when resolved under nonreducing conditions, whereas the protein is entirely monomeric after reduction with 0.1 M DTT. b, Comparison of the Cys mutants resolved under nonreducing conditions. c, An ero1-1 strain (CKY598) was transformed with the control plasmid, pRS316, with PGAL1-ERV2 or with PGAL1-ERV2 bearing Cys mutations. The failure of the Cys mutants to suppress the growth defect of ero1-1 was shown by incubation at 37 °C for two days on minimal medium with 2% (w/v) galactose. Full Figure and legend (76K)

Although Erv2p is the only member of the family with a Cys-X-Cys motif, a similar disulfide shuttle may occur between Cys pairs in other homologs. Sequence alignments reveal that the quiescin family has a second conserved Cys-X-X-Cys sequence¹⁴ in a position comparable to the Cys-Gly-Cys of Erv2p. Furthermore, Erv1p, like Erv2p, has been observed to form disulfide bonded dimers⁹. Erv1p and ALR have second Cys-X-X-Cys motifs N-terminal, rather than C-terminal, to the primary Cys-X-X-Cys active site. The N-terminal region of Erv1p⁹, but not that of Erv2p⁸, is necessary for the formation of dimers. In Erv1p, 50 residues separate the N-terminal auxiliary Cys-Arg-Ser-Cys sequence from the beginning of helix 1. These 50 residues are rich in Gly and Ser and are predicted to have little secondary structure, indicating that they could easily span the 30 Å between the active site of the opposite protomer and the N-terminus of helix 1. Although Ero1p is not homologous to Erv2p and its structure is unknown, the Ero1 protein may operate by a similar disulfide-shuttle mechanism. Ero1p has two pairs of conserved Cys residues, one of which interacts directly with PDI and the other of which reoxidizes the PDI-interacting pair³⁴.

Much effort has focused on understanding the role of the residues in the immediate vicinity of the active site Cys residues on the redox properties of thiol:disulfide oxidoreductases³⁵. However, attention is now turning to issues such as steric exclusion of substrates by the tertiary and quaternary structural contexts of Cys-X_n-Cys motifs. For example, the bacterial periplasmic protein DsbC, which normally acts in reduced form to isomerize disulfides and avoids oxidation by the DsbB protein, instead becomes oxidized by DsbB and in turn acts as a disulfide oxidase when mutated to destroy its dimerization interface³⁶. No changes in the vicinity of the active site were necessary. Furthermore, two thioredoxin folds may not be able to interact with one another, for steric reasons, to accomplish intermolecular dithiol-disulfide exchange³⁷. Although the Erv2p active site disulfide is not in a thioredoxin fold, it is at the N-terminus of a helix, and its local geometry is similar to that of the DsbA and thioredoxin active sites (r.m.s. deviation 0.25 Å for Cys-X-X-Cys backbone atoms). Erv2p may naturally oxidize ER enzymes with thioredoxin folds⁶, and substrates that are sterically unable to access the Cys-Gly-Glu-Cys region of Erv2p may instead interact with the Cys-Gly-Cys in the C-terminal tail. The flexibility of the region containing Cys 176 and Cys 178 in Erv2p, as indicated by the protease sensitivity of the polypeptide chain at neighboring residues and the multiple conformations observed crystallographically, may further facilitate transfer of oxidizing equivalents to substrate dithiols. Erv2p can also oxidize lysozyme, a test substrate lacking a thioredoxin fold⁸; whether the C-terminal Cys pair is required for this activity remains to be determined. In general, the structure and redox properties of adaptor Cys-X_n-Cys motifs could tailor the thiol oxidase of the Erv module activity to the particular substrates and physiological functions of each enzyme in the fold family.

The ERV2 coding region, lacking the first 29 amino acids, was inserted into the pET21b(+) (Novagen) expression vector. The resulting Erv2-N construct was transformed into BL21(DE3) plysS (Novagen) cells. Single colonies were inoculated either into M9-ZB medium containing 4% (w/v) glucose or into LB. All cultures contained 100 mg l^-1 ampicillin and 30 mg l^-1 chloramphenicol. Cells were grown at 37 °C to an optical density of A₆₀₀ = 0.7−0.9, isopropyl--d-thiogalactoside (IPTG) and FAD were added to a final concentration of 0.5 mM and 10 M, respectively, and the culture was shifted to 30 °C for a further 3−6 h. Cells were harvested by centrifugation and lysed by sonication in lysis buffer (20 mM KPO₄, pH 7.4, 500 mM NaCl and 10 mM -mercaptoethanol) supplemented with 50 M FAD. Cell debris and membranes were sedimented by centrifugation, and the supernatant was applied at 4 °C to nickel affinity beads (Qiagen) preequilibrated with lysis buffer. After shaking gently overnight, the mixture was loaded onto a column and washed with 10 column volumes lysis buffer supplemented with 25 mM imidazole. Protein was eluted with 10 ml of 20 mM KPO₄, pH 7.4, 500 mM NaCl, 10 mM -mercaptoethanol and 300 mM imidazole. The protein was further purified by hydroxyapatite chromatography eluted with increasing phosphate. Fractions containing Erv2-N were then pooled and dialyzed against 25 mM NaCl and 10 mM Tris, pH 8.0. To obtain Erv2-c, the dialyzed protein was incubated with thermolysin (10 g enzyme per 0.7 mg of protein) for 30 min at room temperature, and the protease-resistant domain was further purified by DEAE chromatography. The eluted protein was pooled and dialyzed against 25 mM NaCl and 10 mM Tris, pH 8.0, and concentrated by centricon (Amicon) to 19 mg ml^-1, as determined spectroscopically in 6 M guanidine-HCl and 10 mM NaPO₄, pH 6.8, assuming an extinction coefficient of 21,980 at 280 nm.

CD was performed on an Aviv model 202 spectrapolarimeter. Protein was dialyzed against a solution containing 2 mM citrate, pH 6.1, 2 mM KPO₄, pH 6.8, 2 mM boric acid and 10 mM NaCl. Samples were diluted in the same buffer such that the absorbance at 454 nm was identical for Erv2-N and Erv2-c and corresponded to 10 M FAD. Spectra were recorded in a 1 mM pathlength cuvette at 25 °C.

Small yellow crystals (0.07 0.07 0.07 mm³) were grown from the Erv2-N preparation by hanging drop vapor diffusion over a well containing 0.1 M cacodylic acid, pH 6.2, 50 mM MgCl₂ and 32% (w/v) PEG 8000. For Erv2-c, initial crystals of poor morphology were grown over a well solution containing 0.1 M cacodylic acid, pH 6.2, 10 mM CoCl₂ and 22% (w/v) PEG 8000. Large crystals could be grown by seeding from these crystals into a variety of conditions. Crystals for data collection were grown in 0.1 M cacodylic acid, pH 6.2, 10% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) glycerol and 10% (w/v) PEG 1000. Crystals were transferred to a 1:1 mixture of mineral oil and Parabar oil (Exxon) before flash freezing. Diffraction data were collected to 2.0 Å (Erv2-N preparation) and 1.5 Å (Erv2-c) resolution at 120 K on an RU-H3R generator (Rigaku, Tokyo) equipped with a RaxisIV detector (Rigaku) and osmic mirrors. Phasing was performed by MAD using a SeMet derivative of Erv2-c prepared according to published protocol³⁸, except that 10 M FAD was added to the media at the time of induction, and the cells were allowed to grow for 3 h more before harvesting. SeMet-containing crystals were grown under similar conditions to the native crystals. Data for phasing were collected at 100 K on the ESRF ID14 4 beamline at three wavelengths around the selenium absorbtion edge (Table 1). All native and MAD data were processed and scaled using DENZO and SCALEPACK³⁹. Heavy atoms sites were located, and phasing was performed with SOLVE⁴⁰. The noncrystallographic symmetry operator for the Erv2-c crystals was determined using polarrfn⁴¹ and GETAX⁴². The Erv2-c structure was built using O⁴³, and an Erv2-c dimer was used as the molecular replacement search model in AMoRe⁴⁴ to provide phases for the cleaved Erv2-N crystals. Structure refinement was done using the Crystallography and NMR System⁴⁵ without noncrystallographic symmetry restraints. A comparison of the Erv2p active site disulfide with Cys-X-X-Cys motifs in proteins of known structure was accomplished using SPASM⁴⁶.

Table 1. Crystallographic and refinement statistics Full Table

Coordinates have been deposited in the Protein Data Bank (accession codes 1JR8 for Erv2-c and 1JRA for Erv2-N).

	Top

1. Frand, A.R. & Kaiser, C.A. The ERO1 gene of yeast is required for oxidation of protein dithiols in the endoplasmic reticulum. Mol. Cell 1, 161−170 (1998). | Article | PubMed | ISI | ChemPort |
2. Pollard, M.G., Travers, K.J. & Weissman, J.S. Ero1p: a novel and ubiquitous protein with an essential role in oxidative protein folding in the endoplasmic reticulum. Mol. Cell 1, 171−182 (1998). | Article | PubMed | ISI | ChemPort |
3. Tu, B.P., Ho-Schleyer, S.C., Travers, K.J. & Weissman, J.S. Biochemical basis of oxidative protein folding in the endoplasmic reticulum. Science 290, 1571−1574 (2000). | Article | PubMed | ISI | ChemPort |
4. Frand, A.R. & Kaiser, C.A. Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum. Mol. Cell 4, 469−477 (1999). | Article | PubMed | ISI | ChemPort |
5. Noiva, R. Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum. Semin. Cell Dev. Biol. 10, 481−493 (1999). | Article | PubMed | ISI | ChemPort |
6. Sevier, C.S., Cuozzo, J.W., Vala, A., Aslund, F. & Kaiser, C.A. A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation. Nature Cell Biol. 3, 874−882 (2001). | Article | PubMed | ISI | ChemPort |
7. Stein, G. & Lisowsky, T. Functional comparison of the yeast scERV1 and scERV2 genes. Yeast 14, 171−180 (1998). | Article | PubMed | ISI | ChemPort |
8. Gerber, J., Mühlenhoff, U., Hofhaus, G., Lill, R. & Lisowsky, T. Yeast Erv2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family. J. Biol. Chem. 276, 23486−23491 (2001). | Article | PubMed | ISI | ChemPort |
9. Lee, J.-E., Hofhaus, G. & Lisowsky, T. Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase. FEBS Lett. 477, 62−66 (2000). | Article | PubMed | ISI | ChemPort |
10. Hagiya, M. et al. Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution. Proc. Natl. Acad. Sci. USA 91, 8142−8146 (1994). | PubMed | ChemPort |
11. Senkevich, T.G., White, C.L., Koonin, E.V. & Moss, B. A viral member of the Erv1/ALR protein family paricipates in the cytoplasmic pathway of disulfide bond formation. Proc. Natl. Acad. Sci. USA 97, 12068−12073 (2000). | Article | PubMed | ChemPort |
12. Hoober, K.L., Glynn, N.M., Burnside, J., Coppock, D.L. & Thorpe, C. Homology between egg white sulfhydryl oxidase and quiescin Q6 defines a new class of flavin-linked sulfhydryl oxidases. J. Biol. Chem. 274, 31759−31762 (1999). | Article | PubMed | ISI | ChemPort |
13. Hoober, K.L., Sheasley, S.L., Gilbert, H.F. & Thorpe, C. Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides. J. Biol. Chem. 274, 22147−22150 (1999). | Article | PubMed | ISI | ChemPort |
14. Coppock, D.L., Cina-Poppe, D. & Gilleran, S. The quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: thioredoxin and ERV1. Genomics 54, 460−468 (1998). | Article | PubMed | ISI | ChemPort |
15. Coppock, D., Kopman, C., Gudas, J. & Cina-Poppe, D.A. Regulation of the quiescence-induced genes: quiescin Q6, decorin, and ribosomal protein S29. Biochem. Biophys. Res. Commun. 269, 604−610 (2000). | Article | PubMed | ISI | ChemPort |
16. Fraaije, M. W. & Mattevi, A. Flavoenzymes: diverse catalysts with recurrent features. Trends Biochem. Sci. 25, 126−132 (2000). | Article | PubMed | ISI | ChemPort |
17. Kemmink, J., Darby, N. J., Dijkstra, K., Nilges, M. & Creighton, T.E. Structure determination of the N-terminal thioredoxin-like domain of protein disulfide isomerase using multidimensional heteronuclear 13C/15N NMR spectroscopy. Biochemistry 35, 7684−7691 (1996). | Article | PubMed | ISI | ChemPort |
18. Kemmink, J. et al. The structure in solution of the b domain of protein disulfide isomerase. J. Biomol NMR 13, 357−368 (1999). | Article | PubMed | ISI | ChemPort |
19. Martin, J.L., Bardwell, J.C.A. & Kuriyan, J. Crystal structure of the DsbA protein required for disulphide bond formation in vivo. Nature 365, 464−468 (1993). | Article | PubMed | ISI | ChemPort |
20. McCarthy, A.A. et al. Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli. Nature Struct. Biol. 7, 196−199 (2000). | Article | PubMed | ISI | ChemPort |
21. Massey, V. Introduction: flavoprotein structure and mechanism. FASEB J. 9, 473−475 (1995). | PubMed | ISI | ChemPort |
22. van Berkel, W.J.H., Benen, J.A.E., Eppink, M.H.M & Fraaije, M.W. In Flavoprotein protocols (eds Chapman, S.K. & Reid, G.A.) 61−85 (Humana Press, Totowa; 1999). | ChemPort |
23. Kamtekar, S. & Hecht, M.H. The four-helix bundle: what determines a fold? FASEB J. 9, 1013−1022 (1995). | PubMed | ISI | ChemPort |
24. Holm, L. & Sander, C. Mapping the protein universe. Science 273, 595−602 (1996). | PubMed | ISI | ChemPort |
25. Kuriyan, J. et al. Convergent evolution of similar function in two structurally divergent enzymes. Nature 352, 172−174 (1991). | Article | PubMed | ISI | ChemPort |
26. Schulz, G.E., Schirmer, R.H., Sachsenheimer, W. & Pai, E.F. The structure of the flavoenzyme glutathione reductase. Nature 273, 120−124 (1978). | PubMed | ISI | ChemPort |
27. Krishnaswamy, S. & Rossmann, M.G. Structural refinement and analysis of Mengo virus. J. Mol. Biol. 211, 803−844 (1990). | Article | PubMed | ISI | ChemPort |
28. Smith, C.A., Toogood, H.S., Baker, H.M., Daniel, R.M. & Baker, E.N. Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 Å resolution. J. Mol. Biol. 294, 1027−1040 (1999). | Article | PubMed | ISI | ChemPort |
29. Barbirz, S., Jakob, U. & Glocker, M.O. Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone. J. Biol. Chem. 275, 18759−18766 (2000). | Article | PubMed | ISI | ChemPort |
30. Jakob, U., Muse, W., Eser, M. & Bardwell, J.C.A. Chaperone activity with a redox switch. Cell 96, 341−352 (1999). | Article | PubMed | ISI | ChemPort |
31. Graumann, J. et al. Activation of the redox-regulated molecular chaperone Hsp33 — a two-step mechanism. Structure 9, 377−387 (2001). | Article | PubMed | ISI | ChemPort |
32. Zhang, R. & Snyder, G.H. Dependence of formation of small disulfide loops in two-cysteine peptides on the number and types of intervening amino acids. J. Biol. Chem. 31, 18472−18479 (1989).
33. Gleason, F.K., Lim, C.-J., Gerami-Nejad, M. & Fuchs, J.A. Characterization of Escherichia coli thioredoxins with altered active site residues. Biochemistry 29, 3701−3709 (1990). | Article | PubMed | ISI | ChemPort |
34. Frand, A.R. & Kaiser, C. Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum. Mol. Biol. Cell 11, 2833−2843 (2000). | PubMed | ISI | ChemPort |
35. Chivers, P.T., Prehoda, K.E. & Raines, R.T. The CXXC motif: a rheostat in the active site. Biochemistry 36, 4061−4066 (1997). | Article | PubMed | ISI | ChemPort |
36. Bader, M.W. et al. Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA. EMBO J. 20, 1555−1562 (2001). | Article | PubMed | ChemPort |
37. Katzen, F. & Beckwith, J. Transmembrane electron transfer by the membrane protein DsbD occurs via a disulfide bond cascade. Cell 103, 169−779 (2000). | Article | PubMed |
38. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105−124 (1993). | Article | PubMed | ISI | ChemPort |
39. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307−326 (1997). | Article | PubMed | ISI | ChemPort |
40. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849−861 (1999). | Article | PubMed | ISI |
41. Collaborative computational project, Number 4. The CCP4 suite programs for protein crystallography. Acta Crystallogr. D 50, 760−763 (1994). | Article | ISI |
42. Vonrhein, C. & Schulz, G.E. Locating proper non-crystallographic symmetry in low-resolution electron-density maps with the program GETAX. Acta Crystallogr. D 55, 225−229 (1999). | Article | PubMed | ISI |
43. The O Files (29 November, 2000) http://origo.imsb.au.dk/~mok/o.
44. Navaza, J. AmoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157−163 (1994). | Article | ISI |
45. Brünger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905−921 (1998). | Article | PubMed | ISI |
46. Kleywegt, G.J. Recognition of spatial motifs in protein structures. J. Mol. Biol. 285, 1887−1897 (1999). | Article | PubMed | ISI | ChemPort |
47. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946−950 (1991). | Article | ISI |
48. Carson, M. Ribbons. Methods Enzymol. 277, 493−505 (1997). | Article | ISI | ChemPort |
49. Cowtan, K. A CCP4 density modification package. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34−38 (1994).

	Top