Structural Basis of an ERAD Pathway Mediated by the ER-Resident Protein Disulfide Reductase ERdj5

Abstract

ER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters. Our systematic biochemical analyses indicated that two thioredoxin domains that constitute the C-terminal cluster form the highly reducing platform that interacts with EDEM1 and reduces EDEM1-recruited substrates, leading to their facilitated degradation. The pulse-chase experiment further provided direct evidence for the sequential movement of an ERAD substrate from calnexin to the downstream EDEM1-ERdj5 complex, and then to the retrotranslocation channel, probably through BiP. We present a detailed molecular view of how ERdj5 mediates ERAD in concert with EDEM1.

Redox-assisted regulation of Ca 2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5

“…Using a set of ERdj5 mutants in which only one CXXC motif remained intact, we found that SERCA2b preferentially bound to ERdj5 via Trx3 and Trx4 (Fig. 1D), which have significant reductase activity and are hence involved in ERAD (13). This was more directly shown using the ERdj5/CA4 mutant, in which the CXXC motif in the Trx4 domain was mutated to CXXA and other CXXCs to AXXA.…”

“…Human SERCA2b cDNA was amplified by PCR from the Matchmaker Pretransformed Human HeLa library (Clontech) and subcloned into pcDNA3.1. Mouse ERdj5/WT and other ERdj5 mutants (AA, CA, H63A, C1, C2, C3, and C4) were constructed as described previously (12,13). The SERCA2b C875AC887A (SERCA2b/AA) and R923KR988K (SERCA2b/KK) double mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene).…”

“…DLS Measurement. His-ERdj5/SS was overexpressed in Escherichia coli and purified as described previously (13) and used for DLS measurement. The size distributions of ERdj5/SS were determined using a Malvern Zetasizer Nano ZS (Malvern Instruments) essentially as described previously (38).…”

“…When all CXXC motifs were converted to Ala-X-X-Ala (AXXA) (Fig. 1C), this ERdj5/AA mutant lost its reducing activity (12,13). The ERdj5/AA mutant barely bound to SERCA2b (Fig.…”

“…Misfolded substrates are transferred from ERdj5 to Binding Ig Protein (BiP), a major molecular chaperone in the ER, which binds to ERdj5 through the HPD (Hys-Pro-Asp) motif in the J domain. BiP recruits the substrates to the retrotranslocation channel to promote their ER-associated degradation (ERAD) pathway (13,14).…”