Mapping of a Substrate Binding Site in the Protein Disulfide Isomerase-related Chaperone Wind Based on Protein Function and Crystal Structure

The protein disulfide isomerase (PDI)-related protein Wind is essential in <i>Drosophila melanogaster</i>, and is required for correct targeting of Pipe, an essential Golgi transmembrane 2-<i>O</i>-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 <i>in vitro</i>, and we map an essential substrate binding site in Wind to the vicinity of an exposed cluster of tyrosines within the thioredoxin fold domain. <i>In vitro</i>, 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.