Abstract
22 min readIn the developing Drosophila melanogaster embryo, dorsal-ventral patterning displays an absolute requirement for the product of the essential windbeutel gene, Wind. In homozygous windbeutel mutant flies, dorsal-ventral patterning fails to initiate because of the failure of the Golgi-resident proteoglycan-modifying protein, Pipe, to exit the endoplasmic reticulum, and this leads to the death of the embryo. Here, we describe the three-dimensional structure of Wind at 1.9-Å resolution and identify a candidate surface for interaction with Pipe. This represents the first crystal structure of a eukaryotic protein-disulfide isomerase-related protein of the endoplasmic reticulum to be described. The dimeric protein is composed of an N-terminal thioredoxin domain and a C-terminal α-helical domain unique to protein-disulfide isomerase D proteins. Although Wind carries a CXXC motif that is partially surface accessible, this motif is redox inactive, and the cysteines are not required for the targeting of Pipe to the Golgi. However, both domains are required for targeting Pipe to the Golgi, and, although the mouse homologue ERp28 cannot replace the function of Wind, exchange of the Wind D-domain with that of ERp28 allows for efficient Golgi transport of Pipe. In the developing Drosophila melanogaster embryo, dorsal-ventral patterning displays an absolute requirement for the product of the essential windbeutel gene, Wind. In homozygous windbeutel mutant flies, dorsal-ventral patterning fails to initiate because of the failure of the Golgi-resident proteoglycan-modifying protein, Pipe, to exit the endoplasmic reticulum, and this leads to the death of the embryo. Here, we describe the three-dimensional structure of Wind at 1.9-Å resolution and identify a candidate surface for interaction with Pipe. This represents the first crystal structure of a eukaryotic protein-disulfide isomerase-related protein of the endoplasmic reticulum to be described. The dimeric protein is composed of an N-terminal thioredoxin domain and a C-terminal α-helical domain unique to protein-disulfide isomerase D proteins. Although Wind carries a CXXC motif that is partially surface accessible, this motif is redox inactive, and the cysteines are not required for the targeting of Pipe to the Golgi. However, both domains are required for targeting Pipe to the Golgi, and, although the mouse homologue ERp28 cannot replace the function of Wind, exchange of the Wind D-domain with that of ERp28 allows for efficient Golgi transport of Pipe. Within the developing Drosophila embryo, dorsal-ventral polarity is determined by a group of 11 genes that act to create a gradient of the NFκB homologue Dorsal within the nuclei of the embryo. Dorsal is translocated to the nucleus from the cytoplasm upon activation of the transmembrane Toll receptor by an extracellular ligand, Spätzle (1Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (690) Google Scholar). Three of the eleven genes, i.e. nudel, pipe, and windbeutel, are expressed in the follicle cells. Deletion of any one of these three leads to a lethal impairment in establishing dorsal-ventral polarity (2Sen J. Goltz J.S. Konsolaki M. Schupbach T. Stein D. Development. 2000; 127: 5541-5550Crossref PubMed Google Scholar). The products of pipe and windbeutel, Pipe and Wind, are specifically required in a band of follicle cells on the ventral side of the embryo. Pipe is a Golgi-resident type II transmembrane protein with homology to mammalian heparan sulfate 2-O-sulfotransferase (3Sergeev P. Streit A. Heller A. Steinmann-Zwicky M. Dev. Dyn. 2001; 220: 122-132Crossref PubMed Scopus (20) Google Scholar, 4Kobayashi M. Habuchi H. Yoneda M. Habuchi O. Kimata K. J. Biol. Chem. 1997; 272: 13980-13985Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). It is believed that Pipe modifies an as yet unidentified proteoglycan that later participates in the proteolytic activation of Spätzle in the perivitelline space. windbeutel is expressed in all follicle cells located over the oocyte, although it seems essential only in the ventral layer (5Konsolaki M. Schupbach T. Genes Dev. 1998; 12: 120-131Crossref PubMed Scopus (59) Google Scholar). Wind is an endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; GFP, green fluorescent protein; EGFP, enhanced GFP; PDI, protein-disulfide isomerase; PDI-D, PDI-related protein containing a D-domain; MES, 4-morpholineethanesulfonic acid; PEG, polyethylene glycol.-resident, luminal protein belonging to the protein-disulfide isomerase (PDI) family, a group of proteins with varying redox and chaperone properties (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 7Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar). Although the mechanism of action remains unclear, it has recently been shown that an important role for Wind is the correct targeting of Pipe to the Golgi; windbeutel-deficient female flies show an aberrant distribution of Pipe protein, and, when expressed in COS-7 cells, Pipe is retained in the ER in a presumably inactive form. Upon simultaneous overexpression of Wind, however, a clear redistribution of Pipe to the Golgi was observed (2Sen J. Goltz J.S. Konsolaki M. Schupbach T. Stein D. Development. 2000; 127: 5541-5550Crossref PubMed Google Scholar). The mammalian homologues of Wind, ERp28 (8Ferrari D.M. Nguyen Van P. Kratzin H.D. Soling H.D. Eur. J. Biochem. 1998; 255: 570-579Crossref PubMed Scopus (75) Google Scholar) and ERp29 (9Demmer J. Zhou C. Hubbard M.J. FEBS Lett. 1997; 402: 145-150Crossref PubMed Scopus (73) Google Scholar, 10Mkrtchian S. Fang C. Hellman U. Ingelman-Sundberg M. Eur. J. Biochem. 1998; 251: 304-313Crossref PubMed Scopus (89) Google Scholar), have been found in complexes with secretory proteins (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 11Sargsyan E. Baryshev M. Szekely L. Sharipo A. Mkrtchian S. J. Biol. Chem. 2002; 277: 17009-17015Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These proteins are members of the PDI-D subfamily of PDI-related proteins, homologues of which can be found in yeast and plants as well (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). PDI-D proteins are characterized by the presence of one or two domains of roughly 100–120 residues with sequence similarity to the thioredoxin fold of PDI, followed by a novel, α-helical region at their C terminus termed the D-domain (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). The protein-disulfide isomerases are highly abundant proteins that catalyze the formation, reduction, and isomerization of disulfide bonds in substrate proteins, an activity dependent upon the presence of a redox-active CXXC tetrapeptide (7Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar). For some of these proteins it has been shown that, in addition to their redox/isomerase activities, they can also function as chaperones (12McLaughlin S.H. Bulleid N.J. Biochem. J. 1998; 331: 793-800Crossref PubMed Scopus (45) Google Scholar, 13Cai H. Wang C.C. Tsou C.L. J. Biol. Chem. 1994; 269: 24550-24552Abstract Full Text PDF PubMed Google Scholar, 14Wang L. Fast D.G. Attie A.D. J. Biol. Chem. 1997; 272: 27644-27651Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Gillece P. Luz J.M. Lennarz W.J. de La Cruz F.J. Romisch K. J. Cell Biol. 1999; 147: 1443-1456Crossref PubMed Scopus (154) Google Scholar). Recent evidence has shown that many mammalian PDI-related proteins are composed entirely of thioredoxin domains (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 16Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Whereas redox function requires a CXXC motif, substrate binding requires primarily the redox-inactive b′ domain, although all domains are likely to participate. In yeasts and plants, the thioredoxin domains of PDI-D proteins are redox active (these proteins are therefore termed PDI-Dα proteins) and can rescue the otherwise lethal disruption of the pdi gene in Saccharomyces cerevisiae (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 17Monnat J. Neuhaus E.M. Pop M.S. Ferrari D.M. Kramer B. Soldati T. Mol. Biol. Cell. 2000; 11: 3469-3484Crossref PubMed Scopus (46) Google Scholar). Mammalian homologues, however, have a single, b-type, redox-inactive thioredoxin domain (termed PDI-Dβ proteins) (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). Here, we report on the successful crystal structure determination of a key component of the dorsal-ventral patterning pathway, which is also the first crystal structure of an ER-luminal, PDI-related protein. Through mutagenesis and functional assays, we identify a surface of the protein in the N-terminal b-domain that is likely to interact with Pipe. Replacement of a polar residue within this surface with a basic one severely impedes targeting of Pipe to the Golgi. In contrast, the nearby CXXC motif, which is redox inactive, is not required for Golgi targeting. The N-terminal domain of Wind, when expressed alone, does not promote Pipe transport to the Golgi. Furthermore, we find that although a mouse homologue of Wind, ERp28, cannot target Pipe to the Golgi, the D-domain of ERp28 can efficiently replace that of the fly protein. Thus, although the b-domain contains a site that may be involved in interaction with Pipe, the C-terminal D-domain is also required. Cell Lines, Bacterial Strains, Plasmids, and Expression Vectors— COS-7 cells were purchased from the European Collection of Animal Cell Cultures, XL1-Blue bacterial cells and the pBlueScript II plasmid were from Stratagene, pEGFP-N1 was obtained from Clontech, and the pQE-30 and pQE-60 expression vectors were from Qiagen. Antibodies and Reagents—Antibodies against Drosophila Wind were raised in rabbits against the recombinant, full-length, His6-tagged protein from Escherichia coli according to standard procedure. IgG fractions were isolated over protein A-Sepharose from Amersham Biosciences. Rabbit antibodies against the C-terminal KEEL retrieval sequence were prepared as described elsewhere (18Majoul I.V. Bastiaens P.I.H. Söling H.D. J. Cell Biol. 1996; 133: 777-789Crossref PubMed Scopus (137) Google Scholar). Goat anti-rabbit Cy3-conjugated antibody was from Jackson ImmunoResearch Laboratories. For mutagenesis, the QuikChange site-directed mutagenesis kit from Stratagene was used. Cell culture media were from Sigma, and the Pefabloc SC protease inhibitor was purchased from Biomol. cDNA Cloning and Expression Vector Construction—Drosophila windbeutel cDNA encoding the full-length Wind was amplified by PCR from a λZAP cDNA library (kind gift of Dr. M. Takamori, Max-Planck Institute of Biophysical Chemistry). For bacterial expression, a BamHI/SacI PCR product thereof, encoding mature Wind, was ligated into pQE-30, generating an N-terminal extension including a His6 tag (MRGSHHHHHHGS). This construct is referred to as His-Wind. A further construct harboring C-terminally tagged Wind (Wind-His) was prepared by ligating a PCR-amplified transcript of Wind into the NcoI/BglII sites of pQE-60. Mouse ERp28 cDNA was amplified by reverse transcription PCR from F9 cells. For expression in mammalian cells, full-length windbeutel was incorporated into the EcoRI/BamHI sites of pEGFP-N1 such that a stop codon preceded the enhanced green fluorescent protein (EGFP) sequence, ensuring translation of Wind alone. A similar construct was made for ERp28. Wind point mutants (Wind-STGS, Wind-Y55K, and Wind-D31N) were constructed using appropriate primers and the QuikChange site-directed mutagenesis kit. Truncated versions of Wind encoding the N-terminal thioredoxin domain alone, with or without an appended retrieval sequence (Wind-N and Wind-N-KEEL, respectively), were constructed by PCR such that a stop codon was introduced immediately after Ser137 (for Wind-N) or immediately after an appended 12-bp fragment encoding the KEEL terminal retrieval sequence. A Wind-ERp28 fusion construct (Wind-N-p28D) was prepared by fusing the sequence for the C-terminal domain of mouse ERp28 (residues 149–262; Gln150 replaced with Ser) in-frame with that of the b-domain of Wind (residues 1–137), thereby effectively exchanging the Wind D-domain for that of mouse ERp28. Because of difficulty in obtaining an intact Pipe transcript, pipe was amplified in three parts. A XhoI/XbaI fragment comprising 299 bp of the 5′-end of pipe (fragment 1) and an EcoRI/BamHI fragment (fragment 4) comprising 298 bp of the 3′-end (908–1212 of pipe) were amplified from a Drosophila cDNA clone (RE11403; ResGen). An XbaI/BsmI fragment (fragment 2) of 544 bp covering bases 300–843 of pipe was obtained by PCR amplification of genomic DNA from Oregon-R flies (a kind gift from Dr. M. Takamori, Max-Planck Institute for Biophysical Chemistry). All fragments were ligated into pBlueScript II. A final fragment of 64 bp (fragment 3) linking fragments 2 and 4 (bases 844–907 of pipe) was synthesized chemically (MWG Biotech) and used to join fragments 2 and 4 via a BsmI and an introduced MluI site. The MluI site was later reverted to the original sequence to generate full-length wild-type pipe (1212 bp). Pipe-GFP was constructed by ligating a KpnI/BamHI fragment from cloned Pipe into pEGFP-N1. Expression from this construct generates a translocation-competent, full-length Pipe protein fused inframe to the N terminus of the EGFP. Cell Culture and Immunofluorescence Assays—COS-7 cells were grown in 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum with 2 mm l-glutamine and antibiotics at 37 °C and 10% CO2. For immunofluorescence labeling, cells were transfected by electroporation in a Gene Pulser II device (Bio-Rad) and grown on coverslips overnight (20 h) before fixation with 4% paraformaldehyde. Wind antibody was used at a 1:150 dilution, and detection of bound antibody was performed with a 1:1000 dilution of a goat anti-rabbit Cy3-conjugated antibody. Visualization of Cy3 and GFP was carried out on an Axiovert 200 microscope (Zeiss) with filter sets with an excitation filter of 565/30 nm and 480/40 nm, a dichroic beam splitter of 595 nm and 505 nm, and emission filters of 645/75 nm and 527/30 nm, respectively. Protein Expression and Purification—Wild-type Wind protein as well as Wind-Y55K and Wind-D31N were expressed from pQE-His-Wind and pQE-Wind-His in E. coli XL1-Blue cells by induction of an OD600 = 0.7 culture for 3 h at 37 °C with 1 mm isopropyl-1-thio-β-d-galactopyranoside. The recombinant proteins were harvested by brief sonication of lysozyme-treated cells in pH 8-adjusted phosphate buffered saline including 0.2 mm Pefabloc SC protease inhibitor followed by addition of Triton X-100 to 0.1% (v/v) and gel filtration over a nickel-nitrilotriacetic acid nickel affinity column (Qiagen). Bound protein was washed with 4 bed volumes each of wash buffer (20 mm Tris-Cl, pH 8.0, 150 mm NaCl, and 0.1% (v/v) Triton X-100) and salt wash buffer (20 mm Tris-Cl, 350 mm NaCl, and 0.1% Triton X-100), then washed again with 4 bed volumes of pre-elution buffer (20 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.01% Triton X-100, and 8 mm imidazole) and eluted in 4 bed volumes of elution buffer (20 mm Tris-Cl, pH 8.0, 300 mm NaCl, 100 mm imidazole, and 0.01% Triton X-100). Eluted protein was dialyzed extensively against dialysis buffer (10 mm HEPES, pH 7.5, 50 mm NaCl, and 0.01% (v/v) 2-mercaptoethanol), concentrated to 20–25 mg/ml, and stored at 4 °C. Protein purity was verified by 12.5% SDS-PAGE and was consistently >95% pure (for wild-type and all mutants). Size Determination of Recombinant Protein—To estimate the molecular mass of recombinant Wind monomers/oligomers, multi-angle light scattering (19Wyatt P.J. Anal. Chim. Acta. 1993; 272: 1-40Crossref Scopus (1299) Google Scholar) on a DAWN DSP MALS photometer and an Optilab DSP interferometric refractometer (Wyatt Technologies) of protein separated by size exclusion chromatography on a Superdex 200 HR column (Amersham Biosciences) was performed. The protein was loaded and run in 30 mm Tris-Cl, pH 7.9, 350 mm NaCl, and 0.01% (v/v) Triton X-100 at room temperature. Results of three runs of 0.28 mg of protein each were averaged for each recombinant species. Crystallization, Cryo Conditions, and Heavy Atom Derivatives— Crystals were grown by the hanging drop vapor diffusion method at 20 °C. The best crystals were grown by mixing 6 μl of 5.8 mg/ml Wind in 5 mm HEPES, pH 7.5, 25 mm NaCl, 0.0025% (v/v) β-mercaptoethanol with a 3-μl reservoir solution containing 0.1 m MES, pH 6.1, 0.1 m CsCl, 2 mm CaCl2, and 16% (v/v) PEG 300. 0.1 m MES, pH 6.1, 0.1 m CsCl, 20% (v/v) PEG300, and 10% (v/v) glycerol was used as the cryo-protectant solution. The mercury derivative was prepared by growing crystals with a well solution of 0.1 m MES, pH 6.1, 0.08 m NaCl, 2 mm CaCl2, and 16% (v/v) PEG 300 and soaking them in 0.1 m MES, pH 6.1, 0.1 m NaCl, 20% (v/v) PEG 300, 10% (v/v) glycerol, and 0.05 mm HgCl2 for 2 weeks. Data Collection and Processing—Three data sets were collected at low temperature (Table I). The data were processed by DENZO (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), SCALEPACK (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), XPREP (Bruker AXS, Madison, WI) and CCP4 (21Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The crystals belong to space group C2, with two molecules in the asymmetric unit. The native data were collected on a MarCCD detector at the synchrotron beamline X11 in Hamburg at the EMBL outstation DESY. The mercury derivative and native in-house data were used for substructure determination.Table IStatistics of the data collection and structure solution and refinementNativeNativeMercury derivativeData CollectionWavelength (Å)0.8111.54181.5418X-ray sourceX11 beamline, EMBL/DESYCopper rotating anodeCopper rotating anodeDetectorMarCCDMar345Mar345SpacegroupC2C2C2Cell parameters (Å or °)a106.678108.107107.998b50.35850.80250.935c98.61699.03099.122β112.84112.45112.77Resolution limits (Å)37.64-1.90 (2-1.90)19.91-2.69 (2.80-2.69)53.23-2.99 (3.10-2.99)Reflections (unique)38,21813,89010,142Completeness (%)99.8 (99.6)99.0 (93.7)99.1 (93.8)Mean I/σ13.96 (4.77)21.19 (3.64)19.29 (6.45)Redundancy4.50 (3.79)7.09 (4.52)6.75 (5.13)R int(%)aR int = Σ|F o2 - F o2 (mean)/Σ|[F o2].5.65 (27.59)7.12 (44.08)6.13 (23.99)R sigma(%)bR sigma = Σ[σ (F o2)]/Σ[F o2].4.47 (19.59)3.88 (29)4.28 (15.50)Refinement statisticsReflections38216R work/R free (%)c5% reflections are selected in thin shells as test data set.,dR work = Σ||F o| - |F c||/Σ|F o for working data set and R free = Σ||F o - F c||/Σ F o for test data set.21.58/25.57Protein atoms3295Solvent atoms152 water, 1 cesiumMean B value (Å2)37.25R.m.s.d. bond length (Å)0.023R.m.s.d. bond angle (°)2.13a R int = Σ|F o2 - F o2 (mean)/Σ|[F o2].b R sigma = Σ[σ (F o2)]/Σ[F o2].c 5% reflections are selected in thin shells as test data set.d R work = Σ||F o| - |F c||/Σ|F o for working data set and R free = Σ||F o - F c||/Σ F o for test data set. Open table in a new tab Structure Solution, Refinement, and Validation—The structure was solved by single isomorphous replacement including anomalous scattering information (SIRAS). The substructure of three mercury atoms was solved and refined by a combination of difference Patterson analysis and direct methods by dual-space recycling using the program SHELXD (22Sheldrick G.M. Hauptman H.A. Weeks C.M. Miller M. Usón I. Arnold E. Rossmann M.G. International Tables for Crystallography. Vol. F. 2001: 333-351Google Scholar) with the SIRAS data set truncated to 3.2 Å. SHELXE (23Sheldrick G.M. Z. Kristallogr. 2002; 217: 644-650Crossref Scopus (361) Google Scholar) was used for phase calculation, density modification, and phase extension. Subsequently, DM was used to do further density modification by solvent flattening and histogram matching. The model was initially built by hand with the aid of MAID (24Levitt D.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1013-1019Crossref PubMed Scopus (117) Google Scholar). Then, the partial model was extended by wARP (25Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar). During the iterative model-building procedure, SHELXE was used for density modification to improve phases and remove model bias. The structure was refined with REFMAC5 (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) using an F-based maximum likelihood target. Model building was performed with Xfit (27McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar), and only atoms for which electron density was visible were built into the model. The structure was validated with PROCHECK (28Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). All the residues have geometry within the Ramachandran allowed region. The diagrams were produced with DINO (www.dino3d.org), MOLSCRIPT (29Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), and RASTER3D (30Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar). Protein Data Bank Accession Numbers—The coordinates and structure factors have been deposited in the Protein Data Bank with the entry code 1OVN. Overall Crystal Structure—Recombinant Drosophila Wind crystallizes as a homodimer (chains A and B). Each chain is composed of two distinct domains connected by a flexible linker of 11 residues (Fig. 1). The N-terminal b-domain has 118 residues and adopts an α/β fold with the order of secondary structure elements being β1-α1-β2-α2-β3-α3-β4-β5-α4. The strands of the β-sheet, with the exception of β4, have a parallel arrangement, forming a central core of all five strands surrounded by the four α-helices. This fold is characteristic for protein-disulfide isomerase and thioredoxin-related proteins (31Katti S.K. LeMaster D.M. Eklund H. J. Mol. Biol. 1990; 212: 167-184Crossref PubMed Scopus (536) Google Scholar, 32Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (184) Google Scholar, 33Kemmink J. Dijkstra K. Mariani M. Scheek R.M. Penka E. Nilges M. Darby N.J. J. Biomol. NMR. 1999; 13: 357-368Crossref PubMed Scopus (68) Google Scholar). The C-terminal domain is composed of 107 residues and has a five-helix fold, termed the D-domain (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). This structure, which has all helices in anti-parallel arrangement, represents a novel fold unique to PDI-D proteins. The residues in each monomer were numbered from 22 to 257. Residues 24–252 could be modeled for the first monomer and 23–248 for the second. The N-terminal His6 tag sequence (MRGSHHHHHHGS) could not be seen in the density. Oligomeric State—The crystal structure of Wind clearly shows a dimer (Figs. 1b and 3) formed by a head-to-tail arrangement of the N-terminal b-domains (residues 1–118) with no participation by residues within the D-domain. The dimer interface is nearly symmetric and consists primarily of residues to either side of β1 (residues 24–34), residues within and after α1 (residues 37–43), and residues within and after α2 (residues 70–75), which are involved in a mixture of hydrophobic interactions as well as hydrogen bonding. Important contributions to interface interactions from residues within and around β1 and α1 include multiple contacts between the backbone of the conserved (compared with mammalian PDI-D proteins) Gly26 and side-chain atoms of the conserved Asp31 and similar Leu33 residues, further interactions between a side-chain oxygen of Asp31 and backbone atoms of Thr25, and hydrophobic interactions between the Leu33 and Thr25 residues as well as interactions between the similar Val28 and Ser34. The side chain of Arg41 (lysine in ERp28/29) is involved in a series of hydrophobic contacts with Phe42 and Pro43 as well as with backbone atoms of Lys74 and the conserved Asp75 in the turn after α2. In addition, five water-mediated hydrogen bonds can be detected for residues within and around β1. The buried accessible surface area is ∼758 Å2, which corresponds to ∼6.3% of the surface of the protein. Dimerization creates a deep, hydrophilic cleft, with the approximate dimensions 11 × 11 × 27 Å between the b-domains of the monomers (see Supplementary Fig. 1 in the on-line version of this article). The cleft (hereafter referred to as the dimer cleft), which is rich in negatively charged residues, is flanked by residues from the loop between β2 and α2 and residues from the end of β3 and the following loop, with residues within and around β1 (residues 24–34) at its base. Interestingly, the CTGC motif lies at the floor near the entrance to the dimer cleft (Figs. 1 and 3), and may thus be positioned for reacting with substrate cysteines within the cleft. An electron density synthesis calculated with the derivative data shows clearly defined mercury positions (data not shown) not only for the linker cysteine of either chain but also for the first cysteine of the CTGC motif of one of the two chains (Fig. 1b), suggesting that these sites are freely accessible. Superposition of the structures of the individual domains of the two chains shows excellent correlation of their three-dimensional structures with root mean square deviation values for Cα coordinates of 0.61 and 0.92 Å for the b-domain and the D-domain, respectively. The difference in B-factors of 26.9 Å2 between corresponding Cα atoms of the D-domains (compared with a value of 5.6 Å2 between the b-domains) indicates considerable mobility within the D-domain. Interestingly, the two domains of both chains could not be superimposed simultaneously, reflecting a ∼32° relative displacement of the D-domains, which could be traced to the linker region connecting the N- and C-terminal domains and primarily to rotation around the Ile144 and Gly145 residues. One consequence of this displacement is that, in the crystal, the D-domain rotates in a manner such that the gap between the tip of the D-domain (measured from Cα of Glu-226) of chain B moves from a distance of 20.6 Å to within 9 Å of the dimer cleft (measured from Glu-88). Dimerization of Wind is also supported by multi-angle light scattering (19Wyatt P.J. Anal. Chim. Acta. 1993; 272: 1-40Crossref Scopus (1299) Google Scholar) measurements following size exclusion chromatography (Fig. 2), in vitro and in vivo cross-linking, and native gel analysis (data not shown) of the purified recombinant His6-tagged protein, which suggest a dimer of 2 × 27-kDa apparent molecular mass. The Surface above the Dimer Cleft May Be Involved in Interaction with Pipe, but Wind Dimerization May Not Be Required for Pipe Processing—Association of the monomeric subunits of Wind into the homodimer creates an electrostatically negative, elongated cleft wide enough to partially accommodate an extended polypeptide, with possible further interactions on the dimer surface surrounding the cleft and on juxtaposed regions of the D-domains. The surface around the cleft, which includes a loose cluster of three tyrosines (Tyr53, Tyr55, and Tyr86), shows considerable conservation in PDI-Dβ homologues (Figs. 3 and 4), suggesting perhaps the presence of a common substrate-binding site. This site is close to a cis-proline conserved among PDI-related proteins (Fig. 5).Fig. 5Stereo view of the electron density of Phe105-Ser107. In Wind, Pro106 (P106) assumes a cis conformation, which is typical for corresponding residues in redox-active thioredoxin domains. S107, Ser107; F105, Phe105.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate whether residues around the dimer cleft may be involved in interaction with Pipe, we co-expressed Pipe and Wind in COS-7 cells in which Wind was either expressed as wild-type or with a mutation within the loop before α2 (Y55K). Tyr55 is surface exposed at the verge of the dimer cleft (Fig. 3) but is at least 14 Å distant from the dimer interface. Replacement with lysine was expected to disrupt the electrostatically negative surface of the dimer cleft (Fig. 3b) without affecting the folding of the protein. Pipe failed to exit the ER when co-expressed with Wind-Y55K (Fig. 6j), although the Y55K mutant is highly expressed and retained in the ER (Fig. 6, h and i), indicating that the conserved surface around Tyr55 and/or the surface of the dimer cleft formed by the b-domains is required for Pipe export from the ER. We can exclude the possibility that the Y55K phenotype resulted from an impaired ability of Wind to dimerize, because multi-angle light scattering assays showed a sharp peak corresponding to 57 kDa, which is virtually identical to wild-type Wind (Fig. 2). Furthermore
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