Conserved Structural and Functional Properties of D-domain Containing Redox-active and -inactive Protein Disulfide Isomerase-related Protein Chaperones
2007; Elsevier BV; Volume: 282; Issue: 15 Linguagem: Inglês
10.1074/jbc.m604440200
ISSN1083-351X
AutoresUndine Lippert, Daojun Diao, Naomi Barak, David M. Ferrari,
Tópico(s)Redox biology and oxidative stress
ResumoThe structure and mode of binding of the endoplasmic reticulum protein disulfide isomerase-related proteins to their substrates is currently a focus of intensive research. We have recently determined the crystal structure of the Drosophila melanogaster protein disulfide isomerase-related protein Wind and have described two essential substrate binding sites within the protein, one within the thioredoxin b-domain and another within the C-terminal D-domain. Although a mammalian ortholog of Wind (ERp29/28) is known, conflicting interpretations of its structure and putative function have been postulated. Here, we have provided evidence indicating that ERp29 is indeed similar in both structure and function to its Drosophila ortholog. Using a site-directed mutagenesis approach, we have demonstrated that homodimerization of the b-domains is significantly reduced in vitro upon replacement of key residues at the predicted dimerization interface. Investigation of Wind-ERp29 fusion constructs showed that mutants of the D-domain of ERp29 prevent transport of a substrate protein (Pipe) in a manner consistent with the presence of a discrete, conserved peptide binding site in the D-domain. Finally, we have highlighted the general applicability of these findings by showing that the D-domain of a redox-active disulfide isomerase, from the slime mold Dictyostelium discoideum, can also functionally replace the Wind D-domain in vivo. The structure and mode of binding of the endoplasmic reticulum protein disulfide isomerase-related proteins to their substrates is currently a focus of intensive research. We have recently determined the crystal structure of the Drosophila melanogaster protein disulfide isomerase-related protein Wind and have described two essential substrate binding sites within the protein, one within the thioredoxin b-domain and another within the C-terminal D-domain. Although a mammalian ortholog of Wind (ERp29/28) is known, conflicting interpretations of its structure and putative function have been postulated. Here, we have provided evidence indicating that ERp29 is indeed similar in both structure and function to its Drosophila ortholog. Using a site-directed mutagenesis approach, we have demonstrated that homodimerization of the b-domains is significantly reduced in vitro upon replacement of key residues at the predicted dimerization interface. Investigation of Wind-ERp29 fusion constructs showed that mutants of the D-domain of ERp29 prevent transport of a substrate protein (Pipe) in a manner consistent with the presence of a discrete, conserved peptide binding site in the D-domain. Finally, we have highlighted the general applicability of these findings by showing that the D-domain of a redox-active disulfide isomerase, from the slime mold Dictyostelium discoideum, can also functionally replace the Wind D-domain in vivo. Within the endoplasmic reticulum (ER), 3The abbreviations and trivial names used are: ER, endoplasmic reticulum; PDI, protein disulfide isomerase; PDI-Dα, D-domain containing PDI-related protein with a redox active a-type thioredoxin domain; PDI-Dβ, D-domain containing PDI-related protein with a redox inactive b-type thioredoxin domain; WT, wild type; GFP, green fluorescent protein; EGFP, enhanced GFP. an optimal protein folding environment (1Anken E. Braakman I. Craig E. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 191-228Crossref PubMed Scopus (173) Google Scholar) essential for many secretory proteins is provided by a battery of ER chaperones and folding factors, including the chaperones calnexin/calreticulin (2Trombetta E.S. Parodi A.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 649-676Crossref PubMed Scopus (365) Google Scholar), the Hsp70 homolog immunoglobulin heavy chain binding protein (BiP), and the protein disulfide isomerase (PDI)-related family of redox enzymes and chaperones (3Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (448) Google Scholar, 4Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (171) Google Scholar). PDI-related proteins have various functions (5Ellgaard L. Ruddock L.W. EMBO Rep. 2005; 6: 28-32Crossref PubMed Scopus (635) Google Scholar), including redox and chaperone activities, regulation of calcium homeostasis, and regulation of protein export from the ER for degradation. Although relatively much is known about the respective mechanisms of chaperone action of the calnexin/calreticulin proteins and BiP (6Kleizen B. Braakman I. Curr. Opin. Cell Biol. 2004; 16: 343-349Crossref PubMed Scopus (371) Google Scholar, 7Molinari M. Eriksson K.K. Calanca V. Galli C. Cresswell P. Michalak M. Helenius A. Mol. Cell. 2004; 13: 125-135Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 8Mayer M. Reinstein J. Buchner J. J. Mol. Biol. 2003; 330: 137-144Crossref PubMed Scopus (35) Google Scholar, 9Gething M.J. Semin. Cell Dev. Biol. 1999; 10: 465-472Crossref PubMed Scopus (444) Google Scholar), less is known about the detailed molecular basis of PDI-chaperone activity. This is, to a significant extent, due to the weak character of PDI-substrate interactions. Thus, although it is now known that not only the chaperone function of these proteins but to varying extents their redox activity as well relies on their non-covalent interaction with substrate proteins (4Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (171) Google Scholar, 10Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 11725-11735Crossref PubMed Scopus (143) Google Scholar, 11Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar, 12Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (298) Google Scholar, 13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), our knowledge of peptide specificity, peptide binding sites, or the molecular basis for the observed substrate selectivity of these proteins remains sketchy. Redox activity and chaperone function of PDI proteins requires interaction with substrates. The principle peptide binding site in PDI has been mapped to the b′-domain, although contributions from other domains occur (12Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (298) Google Scholar). Thus, much attention has been focused on the redox-inactive domains. Recently, peptide binding sites in the b-domains of PDI, ERp57, and the ERp29 ortholog Wind have been described (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 15Pirneskoski A. Klappa P. Lobell M. Williamson R.A. Byrne L. Alanen H.I. Salo K.E.H. Kivirikko K.I. Freedman R.B. Ruddock L.W. J. Biol. Chem. 2004; 279: 10374-10381Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 16Russell S.J. Ruddock L.W. Salo K.E.H. Oliver J.D. Roebuck Q.P. Llewellyn D.H. Roderick H.L. Koivunen P. Myllyharju J. High S. J. Biol. Chem. 2004; 279: 18861-18869Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Interestingly, these binding sites all map to a region close to what would be the catalytically active redox-active sites in the a and a′ domains of PDI. The first crystal structure of a complete PDI family member of the eukaryotic ER was determined in 2003 (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), allowing a direct structural interpretation of the peptide-binding sites. This Drosophila melanogaster protein, Wind, contains an N-terminal thioredoxin b-type domain found in most PDI-related proteins as well as a unique C-terminal domain (the D-domain) found only in the PDI-D group of PDI-related proteins (3Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (448) Google Scholar). The PDI-D proteins comprise two subgroups. PDI-Dα proteins contain one or more redox-active a-type thioredoxin domains, whereas only redox-inactive b-type domains are present in PDI-Dβ proteins. Current knowledge suggests that PDI-Dβ proteins possess chaperone-like activities. Wind is required for export from the ER of an essential Golgi transmembrane proteoglycan-modifying enzyme, Pipe (a 2-O-sulfotransferase), which otherwise remains trapped in the ER (8Mayer M. Reinstein J. Buchner J. J. Mol. Biol. 2003; 330: 137-144Crossref PubMed Scopus (35) Google Scholar), and human and rat ERp29 (the human protein is also known as ERp28) interact with mutant hepatitis B surface antigen and thyroglobulin, respectively (17Ferrari D.M. Nguyen Van P. Kratzin H.D. Söling H.D. Eur. J. Biochem. 1998; 255: 570-579Crossref PubMed Scopus (75) Google Scholar, 18Sargsyan 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). Interestingly, in D. melanogaster Wind, it was shown that substrate-processing activity requires both the b- and D-domains, and a putative peptide binding site has been mapped to each of these domains (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). This identified for the first time, in mammalian PDI-related proteins, a putative peptide-binding site in a domain not related to thioredoxin. Although a mammalian Wind ortholog ERp28/29 cannot replace Wind in Pipe processing, the D-domains of both proteins could be exchanged, indicating functional conservation between the proteins (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The findings on the Wind protein structure and function are an important milestone in the elucidation of PDI and PDI-D chaperone-substrate interaction mechanisms. However, the general applicability of the findings was unclear, as variant dimerization models for the b-domain and tertiary structures of the D-domains of mammalian orthologs had been suggested (19Liepinsh E. Baryshev M. Sharipo A. Ingelman-Sundberg M. Otting G. Mkrtchian S. Structure (Lond.). 2001; 9: 457-471Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 20Hermann V.M. Cutfield J.F. Hubbard M.J. J. Biol. Chem. 2005; 280: 13529-13537Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Mkrtchian and colleagues (19Liepinsh E. Baryshev M. Sharipo A. Ingelman-Sundberg M. Otting G. Mkrtchian S. Structure (Lond.). 2001; 9: 457-471Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) report NMR structures of the individual b- and D-domains of rat ERp29 and claim that residues Asp-71, Phe-118, Arg-122, Asp-123, and Trp-144 are involved in dimerization, whereas residues Gly-67, Glu-68, Gly-97, and Asp-98 may be involved in the formation of higher homo-oligomers. This is in conflict with the findings from our group (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), where the residues identified by crystallographic and biochemical analyses in Wind dimer formation are on the opposite face of Wind and include Gly-26, Val-28, Asp-31, and Lys-41. In addition, Pro-116 (a structurally important residue of the thioredoxin fold) in ERp29 was suggested to have a trans-conformation, whereas the corresponding residues in Wind and other PDI proteins clearly have a cis conformation (3Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (448) Google Scholar, 13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Furthermore, the proposed structure of the D-domain of ERp29 deviates dramatically from that of Wind in the relative orientation of helices 8 and 9 to helices 6 and 7, and the cumulative data together with in vitro biophysical analysis of ERp29 were interpreted as indicating variant functional roles of the respective proteins (20Hermann V.M. Cutfield J.F. Hubbard M.J. J. Biol. Chem. 2005; 280: 13529-13537Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We provide data based on in vivo functional assays and in vitro molecular characterization showing similarities in structure and function between mammalian ERp29 and its D. melanogaster ortholog Wind. Homodimerization of the PDI-D protein, based on association of the thioredoxin b-type domains, occurs via a conserved interface and requires the presence of key residues, the replacement of which reduce dimer formation significantly. For substrates such as Pipe, conservation of function is tightly linked to structural integrity of the D-domain and more precisely to the integrity of a discrete, conserved peptide binding site spanning two structural elements. In PDI-Dα proteins, identified in yeast and plants, a different domain structure and distribution exists than in PDI-Dβ proteins. Here, two redox-active a-type thioredoxin domains generally precede the D-domain, and b-type domains are absent. In contrast to PDI-Dβ proteins, PDI-Dα proteins are capable of complementing PDI-deficient yeast (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 21Monnat 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). Furthermore, the PDI-Dα proteins generally lack classical ER retrieval sequences (3Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (448) Google Scholar, 22Monnat J. Hacker U. Geissler H. Rauchenberger R. Neuhaus E.M. Maniak M. Soldati T. FEBS Lett. 1997; 418: 357-362Crossref PubMed Scopus (52) Google Scholar), and the degree of similarity between the two subclasses of protein is low (overall ∼30% identity between human PDI-Dβ and slime mold PDI-Dα). We show that, despite these differences, the D-domain of a PDI-Dα protein is capable of replacing the D-domain of Wind functionally, suggesting common functional properties among all PDI-D proteins, despite significant evolutionary divergence in primary sequence. Cell Lines, Bacterial Strains, Plasmids, and Expression Vectors—Mammalian COS-7 and Vero cell lines were obtained from the European Collection of Animal Cell Cultures. Escherichia coli XL1-Blue bacterial cells was from Stratagene, the pEGFP-N1 expression vector was from Clontech, and pQE-30 and pQE-60 expression vectors were from Qiagen. Antibodies—Antibodies against full-length Drosophila Wind and human ERp29 were raised as described previously (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 17Ferrari D.M. Nguyen Van P. Kratzin H.D. Söling H.D. Eur. J. Biochem. 1998; 255: 570-579Crossref PubMed Scopus (75) Google Scholar). Goat anti-rabbit Cy3-conjugated antibody was from Jackson ImmunoResearch Laboratories. Bacterial Expression Vector Constructs—D. melanogaster windbeutel full-length cDNA was cloned as described previously (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). For human ERp29, BamHI/HindIII PCR products amplified from pDsb-m28 (17Ferrari D.M. Nguyen Van P. Kratzin H.D. Söling H.D. Eur. J. Biochem. 1998; 255: 570-579Crossref PubMed Scopus (75) Google Scholar) encoding mature ERp29 were ligated into pQE-30, generating a construct with an N-terminal His6 tag extension. The ERp29 mutants ERp29-L39D, ERp29-D42N, ERp29-K52S, and the double mutant ERp29-D42N/K52S were made from this construct using standard mutagenesis techniques after recloning into BamHI/NcoI sites of pQE60. Single domain mouse ERp29 D-domain constructs encoding the complete D-domain and three residues of the linker region (residues Gly-155–Cys-159), harboring the mutations ΔE222 (hereafter referred to as D-ΔE222), E222Q (D-E222Q), A224S/R225A (D-A224S/R225A), R225A (D-R225A), K228E (D-K228E), L229S (D-L229S), or CΔ13 (D-CΔ13), respectively, as well as the WT D-domain (D-WT), were prepared as pQE-30 expression constructs via ligation into the KpnI/HindIII sites. For full-length mouse ERp29, sequences encoding the mature fragments of the WT protein and the mutants ERp29-E222Q, ERp29-R225A/G227S, and ERp29-K228E were created by standard mutagenesis techniques and ligated into BamHI/NcoI sites of pQE-60, generating constructs with a C-terminal His6 tag. Mammalian Expression Vector Constructs—Full-length windbeutel and Wind-b-p29D, incorporated into the EcoRI/BamHI sites of pEGFP-N1, with a stop codon preceding the enhanced green fluorescent protein (EGFP) sequence (Wind*-EGFP-N1 and Wind-b-p29D*-EGFP-N1) are as described in Ma et al. (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) (referred to as Wind and Wind-N-p28D, respectively). Wind-b-p29D point mutants (E222Q, R225A, K228E, L229S, and L242K), double mutant A224S/R225A, the deletion mutants ΔE222 and L242K-CΔ13, as well as the ERp29 thioredoxin domain mutant D42N were constructed using appropriate primers and standard site-directed mutagenesis protocols, using Wind*-EGFP-N1 or Wind-b-p29D*-EGFP-N1 as the template. Dictyostelium discoideum Dd-PDI was amplified by PCR from a GFP-Dd-PDI construct (21Monnat 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) and the fragment corresponding to D-domain residues 260–363 fused to the Wind-b cDNA fragment (residues 1–145) generating the vector Wind-b-DdPDI-D*-EGFP-N1. This construct has an additional 6-residue insert between Wind and the D. discoideum Dd-PDI D-domain sequences coding for Glu-Thr, and a 3′ sequence encoding the KEEL retrieval signal. 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 in 10% CO2. Cycloheximide, a protein synthesis inhibitor (Sigma), was applied to the cells 16 h after transfection by electroporation at 0.2 mg/ml for 4 h. For immunofluorescence labeling, the cells were grown on coverslips overnight (16–20 h) and then fixed with 4% paraformaldehyde. Bound Wind antibody, used at a 1:300 dilution, was detected with a 1:1000 dilution of goat anti-rabbit Cy3-conjugated antibody. Visualization of Cy3 and GFP was carried out on an Axiovert 200 microscope (Zeiss) with excitation filters of 565/30 and 480/40 nm, a dichroic beam splitter of 595 and 505 nm, and emission filters of 645/75 and 527/30 nm, respectively. Protein Expression and Purification—All proteins were expressed in XL1-Blue by induction of A600 = 0.7 cultures for 3 h at 37 °C with 1 mm isopropyl-1-thio-β-d-galactopyranoside. Recombinant ERp29 proteins were harvested by sonication of lysozyme-treated cells in pH 8.0 adjusted phosphate-buffered saline or 20 mm Tris-Cl, pH 8.0, 300 mm NaCl, 8 mm imidazole, including 1 mm Pefabloc SC protease inhibitor followed by the addition of Triton X-100 to 0.1% (v/v) and application on a nickel-nitrilotriacetic acid affinity column (Qiagen). Bound protein was washed and eluted as recommended by the manufacturer. Eluted protein was dialyzed against dialysis buffer (10 mm HEPES, pH 7.5, 50 mm NaCl, 1 mm dithiothreitol), concentrated to 15–30 mg/ml, and stored at 4 °C. For far UV circular dichroism measurements, all proteins were further purified by ion exchange chromatography on either Q-Sepharose or S-Sepharose media (GE Healthcare), dependent on protein pI values (data not shown). Protein concentrations were determined with the ProtParam tool (23Gasteiger E. Hoogland C. Gattiker A. Duvaud S. Wilkins M.R. Appel R.D. Bairoch A. Walker J.M. The Proteomics Protocols Handbook. Humana Press, Hatfield, UK2005: 571-607Crossref Google Scholar) using a calculated absorption coefficient at 280 nm. Protein purity was verified by SDS-PAGE. In Vitro Cross-linking of Recombinant Protein—Purified His6-tagged ERp29 and Wind proteins were rebuffered in assay buffer (50 mm sodium borate, pH 7.4, 150 mm NaCl). Cross-linking was performed with 0.2 μg/μl protein by the addition of glutaraldehyde to 1% (w/v) followed by quenching after 2 min with 2 m NaBH4. Samples were precipitated with trichloroacetic acid and analyzed by SDS-PAGE. Biophysical Analysis of ERp29—Far UV circular dichroism spectra were recorded on a Jasco J710 spectrophotometer. All scans at 20 °C were collected as an average of 5–7 scans, whereas temperature gradient scans were repeated 2–3 times. A cell with a path length of 0.1 cm, a scan speed of 50–100 nm/min, a spectral bandwidth of 1.0 nm, and a time constant of 0.5 s for thermal denaturation was used. The maximal high tension voltage was 700 V. All proteins were dialyzed against 20 mm sodium phosphate, pH 7.5, 50 mm NaCl, and 0.01% Triton X-100 before analysis and were used at 11 μm (full-length proteins), 21 μm (single domain construct D-CΔ13), or 19 μm (all other D-domain constructs). Dimerization of Human ERp29—The b-domain of Wind includes a homodimerization surface encompassing 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) (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Here, important stabilizing interactions are mediated by Gly-26, Val-28, Asp-31, and Arg-41. Dimerization is critical for the function of Wind, and mutants of Wind that cannot dimerize fail to support Pipe processing (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Dimerization of Pipe brings the substrate binding site of each b-domain to within ∼13 Å of each other, which may be important for chaperone action. In contrast, for rat ERp29, it has been suggested (19Liepinsh E. Baryshev M. Sharipo A. Ingelman-Sundberg M. Otting G. Mkrtchian S. Structure (Lond.). 2001; 9: 457-471Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) that the dimerization site of rat ERp29 comprises residues Asp-71 (helix α2, corresponds to Wind Glu-60), Phe-118, Arg-122, Asp-123, (within and after strand β4, Wind Ile-108, Lys-112, Gly-113), and Trp-144 (Phe-135 in helix 4 of Wind). The proposed dimerization surfaces are on different faces of the thioredoxin fold. If this were the case, it might also be the reason for the lack of activity of WT ERp29 in the processing of Pipe in our assays (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Clearly, a correct interpretation of the mode of dimerization of ERp29 is important for understanding its function in vivo, and therefore we chose to analyze participation of residues at the dimerization interface via a mutagenesis approach. In Wind, mutation of Val-28 (V28D) or Asp-31 (D31N) leads to a significant reduction in dimerization (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), as expected. If mammalian ERp29 proteins have the same structure, mutation of either of the corresponding residues of the human protein, Leu-39 and Asp-42, could have a similar effect. Indeed, as shown in Fig. 1, the dimer:monomer ratio in glutaraldehyde-cross-linked, recombinant ERp29 is significantly shifted toward the monomer for both the L39D and the D42N mutants (Fig. 1a, lanes 1 and 2, respectively; single arrow) compared with the ratio observed for WT protein (Fig. 1a, lane 4), which is strongly shifted toward dimer formation (double arrow). Under the same conditions, the reduction in the proportion of dimer in favor of the monomer can clearly be seen for Wind-D31N, the Drosophila equivalent of human ERp29-D42N, compared with the WT protein (Fig. 1b, lanes 1 and 2, respectively). For ERp29-K52S, dimerization is also impaired, although to a lesser extent (Fig. 1a, lane 3). We can exclude the possibility that an alternative dimer interface comes into play at altered pH or salt conditions, as our assays were performed under various conditions (pH 6–8, 0–1 m NaCl) with a similar outcome (data not shown). The D-domain of ERp29 Harbors a Peptide-binding Site Similar to That of Wind—We have previously shown that, in Drosophila Wind, five residues of the D-domain (Glu-212, Arg-215, Arg-218, Leu-219, and Leu-232) are essential for Pipe processing. Mutation of either Arg-215, Arg-218, or Leu-232 was sufficient to cause significant reduction in Pipe transport, whereas mutation of Glu-212 and Leu-219 had no significant effect unless combined (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). With the exception of Leu-232, these residues reside on α-helix 8 of the D-domain (Fig. 2). Together with Leu-232 on helix 9, the residues form a tight, surface-exposed cluster on the same face of the protein as the b-domain binding site (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Surrounding residues do not seem to be involved (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 4M. Sevvana, G. M. Sheldrick, and D. M. Ferrari, manuscript in preparation. Interestingly, work by Ma et al. (13Ma Q. Guo C. Barnewitz K. Sheldrick G.M. Söling H.-D. Usón I. Ferrari D.M. J. Biol. Chem. 2003; 278: 44600-44607Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) also shows that the D-domain of ERp29 could complement the corresponding domain in Wind. However, Mkrtchian and co-workers (19Liepinsh E. Baryshev M. Sharipo A. Ingelman-Sundberg M. Otting G. Mkrtchian S. Structure (Lond.). 2001; 9: 457-471Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) have obtained a significantly different fold for the mammalian PDI-Dβ protein by NMR, leading us to ask whether the peptide binding site identified in the Wind protein is conserved in mammalian ERp29. We therefore studied the effects on Pipe protein translocation to the Golgi using 8 different mutants of the mouse ERp29 D-domain, based on the published Wind data (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In COS cells expressing both Pipe-GFP and Wind or one of its mutants, Golgi transport of Pipe signifies productive ER processing and functional integrity of the PDI-D protein. In cells expressing Pipe-GFP alone, the protein remains in the ER (Fig. 3, panel a4), as originally reported by Stein and co-workers (24Sen J. Goltz J.S. Konsolaki M. Schupbach T. Stein D. Development (Camb.). 2000; 127: 5541-5550Crossref PubMed Google Scholar), and relocates to the Golgi upon Wind coexpression (Fig. 3, panels a1–a3). In the chimeric construct of the Wind b-domain and mouse ERp29 D-domain (Wind-b-p29D), mutation of any one of the corresponding residues (E222Q, R225A, K228E, L229S, and L242K) (Figs. 3 and 4) is sufficient to cause disruption of Pipe export to the Golgi. In Wind, no effect of the Wind E212Q mutation on Pipe transport was observed (14Barnewitz K. Guo C. Sevvana M. Ma Q. Sheldrick G.M. Söling H.-D. Ferrari D.M. J. Biol. Chem. 2004; 279: 39829-39837Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), requiring combination with the likewise tolerated L219S mutation for significant effect. In the chimeric construct, however, each mutation alone affects Pipe processing; the effect of the E222Q mutation is weak but visible, with ∼15% of cells clearly showing ER-retained Pipe (Fig. 4, panels c1–c3), and the Wind-b-p9D-L229S mutant causes clear ER retention in at least 60% of the cells (Fig. 3, panels h1–h3). Similar levels (∼60%) of ER-retained Pipe are observed with the mutants K228E and L242K (Fig. 3, panels g1–g3 and i1–i3). The strongest effect is observed with the R225A mutant, which completely prevents ER export of Pipe (Fig. 3, panels e1–e3). The effects seen cannot be ascribed to low p
Referência(s)