Domain Architecture of Protein-disulfide Isomerase Facilitates Its Dual Role as an Oxidase and an Isomerase in Ero1p-mediated Disulfide Formation

Abstract

Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A at the same rate. In contrast, we found that in the context of full-length PDI, there is an asymmetry in the rate of oxidation of the two active sites. This asymmetry is the result of a dual effect: an enhanced rate of oxidation of the second catalytic (A) domain and the substratemediated inhibition of oxidation of the first catalytic (A) domain. The specific order of thioredoxin domains in PDI is important in establishing the asymmetry in the rate of oxidation of the two active sites thus allowing A and A, two thioredoxin domains that are similar in sequence and structure, to serve opposing functional roles as a disulfide isomerase and disulfide oxidase, respectively. These findings reveal how native disulfide folding is accomplished in the endoplasmic reticulum and provide a context for understanding the proliferation of PDI homologs with combinatorial arrangements of thioredoxin domains.Proteins that traverse the secretory pathway typically contain disulfide bonds that are critical for their correct fold and function. In eukaryotes, the endoplasmic reticulum (ER) 2 is the entry point into the secretory pathway and is the cellular compartment where folding and disulfide bond formation occur (1, 2). Disulfides can form spontaneously in vitro in the presence of an oxidizing agent such as molecular oxygen or oxidized glutathione; however, this process is typically slow and inefficient. In vivo, disulfide bond formation is dependent on cellular machinery to catalyze the formation of new disulfides (oxidation) and the rearrangement of non-native disulfides (isomerization). Both oxidation and isomerization are necessary for allowing the full complement of native disulfide bond formation.Protein-disulfide isomerase (PDI), which was identified more than 40 years ago, plays a critical role in promoting native disulfide bond formation in vivo (3). PDI, an essential enzyme with the ability to catalyze both the oxidation of new disulfides and the isomerization of existing disulfides, is composed of four thioredoxin-like domains (4). The first and last domains (referred to as A and AЈ, respectively) contain Cys-x-x-Cys (CxxC) active sites, whereas the two middle domains (referred to as B and BЈ) are catalytically inactive (5, 6). Oxidation involves the transfer of an active site disulfide from PDI to substrate proteins, while isomerization requires the active site cysteines to be in a reduced form so that they can attack non-native disulfides in substrate ...

Mutations in the FAD Binding Domain Cause Stress-induced Misoxidation of the Endoplasmic Reticulum Oxidoreductase Ero1β

“…2), even though the Ero-PDI docking site and the buried FAD binding site are not likely to be in direct contact. This fits with the idea that Ero1-PDI specificity might arise from both the active site cysteines and direct proteinprotein interactions (23) and that structural changes may occur during electron transfer to the Cxx(CxxC) site in Ero1p (32).…”

Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners

“…Another member of the ER family of Trx-like proteins, ERp57, has been implicated in the oxidation of glycosylated substrates maintained in the calnexin/ calreticulin folding cycle (18). Conflicting results exist on whether ERp57 is reoxidized by Ero1-α as well (19)(20)(21). Although some of the other Trx-like ER proteins have been characterized with respect to redox potential and possible substrates, their biological functions and physiological oxidants remain unknown.…”

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

“…This process produces reactive oxygen species (ROS) in stoichiometric amounts to the number of disulfide bonds formed [11]. Disulfide bond formation is random, and incorrect bond pairs must be exchanged for native bonds via PDI-based processes [12]. In addition, reduced glutathione (GSH) acts as a buffer for the redox state of the ER [13].…”