The Highly Dynamic Nature of ERdj5 Is Key to Efficient Elimination of Aberrant Protein Oligomers through ER-Associated Degradation

Abstract

ERdj5, composed of an N-terminal J domain followed by six thioredoxin-like domains, is the largest protein disulfide isomerase family member and functions as an ER-localized disulfide reductase that enhances ER-associated degradation (ERAD). Our previous studies indicated that ERdj5 comprises two regions, the N- and C-terminal clusters, separated by a linker loop and with distinct functional roles in ERAD. We here present a new crystal structure of ERdj5 with a largely different cluster arrangement relative to that in the original crystal structure. Single-molecule observation by high-speed atomic force microscopy visualized rapid cluster movement around the flexible linker loop, indicating the highly dynamic nature of ERdj5 in solution. ERdj5 mutants with a fixed-cluster orientation compromised the ERAD enhancement activity, likely because of less-efficient reduction of aberrantly formed disulfide bonds and prevented substrate transfer in the ERdj5-mediated ERAD pathway. We propose a significant role of ERdj5 conformational dynamics in ERAD of disulfide-linked oligomers.

The endoplasmic reticulum (ER) chaperone BiP is a master regulator of ER functions: Getting by with a little help from ERdj friends

“…This structure also revealed a binding site for EDEM, a component of the ERAD machinery for glycosylated proteins, allowing ERdj5 to provide a physical lumenal link between ER quality control of BiP clients that are often unglycosylated and lectin clients. A second ERdj5 crystallization attempt produced a different three-dimensional arrangement of the Nand C-terminal clusters (73). This suggested more flexibility in the structure than had been anticipated, and single molecule studies revealed rapid movement between the two clusters.…”

“…In addition to possessing an N-terminal J domain, ERdj5 has six thioredoxin (Trx) domains, two of which are enzymatically inactive (Trx-like), and an ER retention sequence. Structural data show that the Trx domains are arranged in two clusters (72,73). Each cluster possesses two Trx domains, but the N-terminal cluster also has the two catalytically inactive domains.…”

How Are Proteins Reduced in the Endoplasmic Reticulum?

“…Mutational inactivation of the two most reducing thioredoxin domains in ERdj5 was subsequently shown to strongly influence the ERAD-enhancing activity of ERdj5 [18] . The efficiency of ERdj5 in catalyzing reduction-mediated ERAD has recently been shown to rely not only on the redox activity of the protein but also on its conformational plasticity [46] .…”

Biochemical characterizations of ER-resident peroxidases, GPx7 and GPx8, reveal their distinct and regulated oxidative activities

“…In the mammalian ER, the reactions are catalyzed by a variety of oxidoreductases which encompass more than 20 members of the protein disulfide isomerase (PDI) family (1)(2)(3)(4)(5) and more than five PDI oxidases including ER oxidoreductin-1α (Ero1α) (6-9) and peroxiredoxin-4 (Prx4) (10)(11)(12). Some PDI family enzymes also engage in disulfide bond reduction to promote protein folding (13), the ER-associated degradation of misfolded proteins (14)(15)(16), or the ER-to-cytosol retrograde translocation of a bacterial cholera toxin and non-enveloped viruses (17)(18)(19). PDI family members typically possess thioredoxin (Trx)-like domains with a CXXC motif at the redox-active site, thereby exerting thiol-disulfide exchange reactions with substrate proteins (1,3).…”