Structure-Function Analysis of the Endoplasmic Reticulum Oxidoreductase TMX3 Reveals Interdomain Stabilization of the N-terminal Redox-active Domain

Abstract

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb, we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb, the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.For many proteins of the secretory pathway and those targeted to the cell surface or secreted, correct folding depends on the formation of native disulfides. The formation of short range disulfide bonds found in some proteins likely requires little catalysis, whereas other proteins rely on the catalyzed formation and rearrangement of disulfides that must be introduced in a nontrivial pattern. The first ER enzyme found to promote these processes was PDI 3 (1). In vitro, PDI can catalyze oxidation of free cysteines as well as reduction and isomerization of incorrect disulfide bonds (2-4). It is now clear that a whole family of PDI-like proteins of almost 20 members exists in the mammalian ER (5, 6).The proteins of the PDI family all contain at least one domain with a thioredoxin fold, where the secondary structure elements are typically arranged as ␤ 1 -␣ 1 -␤ 2 -␣ 2 -␤ 3 -␣ 3 -␤ 4 -␤ 5 -␣ 4 . Thioredoxin-like domains that encompass a reactive CXXC tetrapeptide sequence are classified as a-type domains and otherwise as b-type domains. For instance, PDI contains four thioredoxin-like domains named a, b, b, and a, where a and a are redox-active, and b and b show no such activity. The redoxactive domains generally show a high level of sequence conservation, whereas the b-type domains display comparatively little sequence similarity. The function of many b-type domains remains unknown. It is clear, however, that the b domain in PDI confers substrate binding (7) and that the corresponding domain in ERp57, the closest homolog of PDI, interacts with the lectin chaperones calnexin and calreticulin that bind glycoprotein substrate...

Novel Thioredoxin-related Transmembrane Protein TMX4 Has Reductase Activity

“…The protein is a type I transmembrane protein that functions as an ER membrane reductase and possesses a CPSC motif within a thioredoxin fold that faces the ER lumen. TMX1 and TMX3 are also TMX family proteins and ER membrane isomerases (21)(22)(23)(24). TMX2 contains an SXXC motif in its Trx-like domain and is therefore thought to be inactive in terms of oxidoreductase activity (6).…”

“…TMX3 is most similar to PDI because of the presence of the b and bЈ domains in addition to the catalytic Trx-like domain (21). TMX3 was suggested from in vitro analysis to have isomerase activity in the ER (21,22). TMX (TMX1) has one Trx-like domain with oxidoreductase activity (23,24), and a recent report demonstrated that TMX1 prevents an overexpressed major histocompatibility complex class I heavy chain from being degraded (25).…”

“…TMX2 has an unusual catalytic motif, SNDC instead of CXXC (20) and is thought to be redoxinactive (6). TMX3 is most similar to PDI because of the presence of the b and bЈ domains in addition to the catalytic Trx-like domain (21). TMX3 was suggested from in vitro analysis to have isomerase activity in the ER (21,22).…”

Protein disulfide isomerases: Impact of thapsigargin treatment on their expression in melanoma cell lines

“…Relative to TMX3 and TMX4, whose expression markedly increases with THG concentration in MNT-1 cells, its role is not well clear. The studies reported for TMX3 [21] and TMX4 [22] suggest that they interact with calnexin and ERp57 forming a complex, which cooperatively enable protein folding. Altogether, the results show that SK-MEL-30 is more resistant than MNT-1 to ER stress induced by THG.…”

Yeast Mpd1p Reveals the Structural Diversity of the Protein Disulfide Isomerase Family

“…For example, the a domain of the a-b-b' PDI-like transmembrane protein TMX3 is more stable in the presence than in the absence of the neighboring noncatalytic domains. 13 One may easily imagine that non-catalytic domains could have significant effects on the redox properties of a PDI family oxidoreductase, should these domains differentially stabilize the oxidized and reduced states of the active trx domain.…”