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The structures of the PDI-related protein Wind (with a C-­terminal His6 tag) and the mutants Y53S, Y53F and Y55K have been determined and compared with the wild-type structure with the His6 tag at the N-terminus. All five structures show the same mode of dimerization, showing that this was not an artefact introduced by the nearby N-terminal His6 tag and suggesting that this dimer may also be the biologically active form. Although the mutants Y53S and Y55K completely abrogate transport of the protein Pipe (which appears to be the primary function of Wind in the cell), only subtle differences can be seen in the putative Pipe-binding region. The Pipe binding in the active forms appears to involve hydrophobic interactions between aromatic systems, whereas the inactive mutants may be able to bind more strongly with the help of hydrogen bonds, which could disturb the delicate equilibrium required for effective Pipe transport. [ABSTRACT FROM AUTHOR]

We report on the isolation, sequence and a putative role of a human endoplasmic-reticulum-lumenal protein, ERp28. The protein has the C-terminal retention signal KEEL and localizes to the endoplasmic reticulum (ER) as seen by subcellular fractionation and immunofluorescence studies. The protein has significant sequence similarity to members of the protein disulfide isomerase (PDI) family, although it lacks the thioredoxin box (CGHC) motif. We propose, on the basis of sequence analysis, a model of the domain structure of PDI, representing a significant extension of previously proposed models. Our results are in partial agreement with recently published NMR data [Kemmink, J., Darby, J., Dijkstra, K., Nilges, M. & Creighton, T. E. (1997) Curr. Biol. 7, 239-245] and indicate that PDI contains, in addition to the two thioredoxin folds described in previous models, two thioredoxin folds within the domains previously defined as b and b′. The thioredoxin domain of ERp28 shares a higher degree of similarity with the corresponding active and inactive domains of PDI than with other members of the PDI family, indicating that ERp28 developed from an ancient form of PDI or a PDI precursor. In contrast to Ig-heavy-chain-binding protein, human ERp28 is not induced by metabolic stress (tunicamycin). In in vitro experiments, ERp28 and calnexin precipitate with overexpressed, wild-type hepatitis B small surface antigen and with a mutated ER-retained form. This indicates that ERp28, as calnexin, may be involved in the processing of secretory proteins within the ER. [ABSTRACT FROM AUTHOR]

Abstract: The protein disulfide isomerase (PDI) family member ERp46/endoPDI/thioredoxin domain-containing protein 5 is preferentially expressed in a limited number of tissues, where it may function as a survival factor for nitrosative stress in vivo. It is involved in insulin production as well as in adiponectin signaling and interacts specifically with the redox-regulatory endoplasmic reticulum proteins endoplasmic oxidoreductin 1α (Ero1α) and peroxiredoxin-4. Here, we show that ERp46, although lacking a PDI-like redox-inactive b′-thioredoxin domain with its hydrophobic substrate binding site, is able to bind to a large pool of peptides containing aromatic and basic residues via all three of its catalytic domains (a0, a and a′), though the a0 domain may contain the primary binding site. ERp46, which shows relatively higher activity as a disulfide-reductase than as an oxidase/isomerase in vitro compared to PDI and ERp57, possesses chaperone activity in vivo, a property also shared by the C-terminal a′ domain. A crystal structure of the a′ domain is also presented, offering a view of possible substrate binding sites within catalytic domains of PDI proteins. [Copyright &y& Elsevier]

The protein disulfide isomerase (PDI)-related protein Wind is essential in Drosophila melanogaster, and is required for correct targeting of Pipe, an essential Golgi transmembrane 2-O-sulfotransferase. Apart from a thioredoxin fold domain present in all PDI proteins, Wind also has a unique C-terminal D-domain found only in PDI-D proteins. Here, we show that Pipe processing requires dimeric Wind, which interacts directly with the soluble domain of Pipe in vitro, and we map an essential substrate binding site in Wind to the vicinity of an exposed cluster of tyrosines within the thioredoxin fold domain. In vitro, binding occurs to multiple sites within the Pipe polypeptide and shows specificity for two consecutive aromatic residues. A second site in Wind, formed by a cluster of residues within the D-domain, is likewise required for substrate processing. This domain, expressed separately, impairs Pipe processing by the full-length Wind protein, indicating competitive binding to substrate. Our data represent the most accurate map of a peptide binding site in a PDI-related protein available to date and directly show peptide specificity for a naturally occurring substrate. [ABSTRACT FROM AUTHOR]