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 Within the endoplasmic reticulum (ER), 3 an optimal protein folding environment (1) essential for many secretory proteins is provided by a battery of ER chaperones and folding factors, including the chaperones calnexin/calreticulin (2), the Hsp70 homolog immunoglobulin heavy chain binding protein (BiP), and the protein disulfide isomerase (PDI)-related family of redox enzymes and chaperones (3, 4).PDI-related proteins have various functions (5), 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 (6 -9), 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 (4, 10 -13), 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 bЈ-domain, although contributions from other domains occur (12). 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 (14 -16). Interestingly, these binding sites all map to a region close to what would be the catalytically active redox-active sites in the a and aЈ domains of PDI.The first crystal structure of a complete PDI family member of the eukaryotic ER was determined in 2003 (13), 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 (3).The PDI-D proteins comprise two subgroups. PDI-D␣ proteins contain one or more redox-active a-type thioredoxin domains, whereas only redox-inactive b-type domains are present in PDI-D proteins. Current knowledge suggests that PDI-D proteins possess chaperone-like activities. Wind is required for export from the ER of an essential Golgi trans-* This work was funded in part by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and an Excellence Network of the State of Sachsen Anhalt. T...