Conserved Structural and Functional Properties of D-domain Containing Redox-active and -inactive Protein Disulfide Isomerase-related Protein Chaperones

Abstract

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...

Dimerization of ERp29, a PDI-like Protein, Is Essential for Its Diverse Functions

“…When the LE was incubated with a cross-linker and then immunoblotted with an antibody against ERp29 (ϳ30 kDa), a ϳ60 kDa band appeared, indicating the formation of an ERp29 homodimer ( Figure 1B, cf. lane 2 to 1), consistent with previous reports (Mkrtchian et al, 1998b;Ferrari et al, 1998;Lippert et al, 2007). Similarly, when purified ERp29 NTD (ϳ17 kDa) was incubated with a cross-linker, a ϳ35-kDa species appeared ( Figure 1B, cf.…”

“…Mutation of D42 to alanine presumably disrupted the hydrogen bond, leading consequently to a marked loss of dimer stability. Consistent with our observation that the D42A rat ERp29 mutant failed to dimerize, the corresponding D31N mutant in Wind (Ma et al, 2003) and the D42N mutant of human ERp29 (Lippert et al, 2007) are also significantly defective in dimerization.…”

The C-Terminal Domain of ERp29 Mediates Polyomavirus Binding, Unfolding, and Infection

“…Studies of the ERp29 D. melanogaster orthologue, Wind, have shown that five residues in the CTD of Wind (E212, R215, R218, L219, and L232) contribute to its ability to chaperone the secretory substrate, Pipe, from the ER to the Golgi compartment (1). Furthermore, when fused to the NTD of Wind, the CTD of mouse ERp29 can functionally replace the corresponding Wind domain, and mutations corresponding to the ERp29 CTD residues (E220Q, R223A, K226E, L227S, and L240K) disrupt the chaperone activity of the Wind-ERp29 hybrid (10,11). We therefore asked whether these residues in ERp29 CTD might play a role in mediating PyV unfolding.…”

“…Although the function of the ERp29 CTD (which consists of helices ␣5 to ␣9) has not been established, Wind CTD has been demonstrated to play a critical role in Wind's chaperone activity, possibly serving as a substrate-binding site (1,10). This finding raises the possibility that the ERp29 CTD may similarly play a crucial role during the PyV unfolding reaction.…”

Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29

“…This is especially important as variant dimerization models for the b domain and dramatically different tertiary structures of the D domains of mammalian orthologs compared to Wind have been suggested and have been interpreted as an indication of variant functional roles of the two proteins. [16][17][18] NMR structures of the individual b and D domains of rat ERp29, reported by Liepinsh et al, 16 indicated involvement of residues Asp71, Phe118, Arg122, Asp123, and Trp144 in dimerization. Furthermore, a conserved proline residue, Pro116, found in the a domains of most PDI proteins in cis conformation, where it plays a structurally and possibly functionally important role, was suggested to be in trans conformation in ERp29, indicating gross structural and functional differences in this domain beyond the variant dimerization properties.…”