Conserved structural and functional properties of D-domain containing redox-active and -inactive protein disulfide isomerase-related protein chaperones.

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.