The lectin chaperone calreticulin (CRT) assists the folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). It interacts with ERp57, a thiol-disulfide oxidoreductase that promotes the formation of disulfide bonds in glycoproteins bound by CRT. Here, we investigated the interaction between CRT and ERp57 by using biochemical techniques and NMR spectroscopy. We found that ERp57 binds to the P-domain of calreticulin, an independently folding domain comprising residues 189 -288. Isothermal titration calorimetry showed that the dissociation constant of the CRT(189 -288)͞ERp57 complex is (9.1 ؎ 3.0) ؋ 10 ؊6 M at 8°C. Transverse relaxation-optimized NMR spectroscopy provided data on the thermodynamics and kinetics of the complex formation and on the structure of this 66.5-kDa complex. The NMR measurements yielded a value of (18 ؎ 5) ؋ 10 ؊6 M at 20°C for the dissociation constant and a lower limit for the first-order exchange rate constant of k off > 1,000 s ؊1 at 20°C. Chemical shift mapping showed that interactions with ERp57 occur exclusively through amino acid residues in the polypeptide segment 225-251 of CRT(189 -288), which forms the tip of the hairpin structure of this domain. These results are analyzed with regard to the functional mechanism of the CRT͞ERp57 chaperone system. G lycoprotein folding and quality control in the endoplasmic reticulum (ER) are assisted by two homologous molecular chaperones, calreticulin (CRT) and the membrane-bound calnexin (CNX). CRT and CNX are lectins that interact with monoglucosylated trimming intermediates of N-linked core glycans, cooperating with enzymes involved in the trimming and modification of the glycans (1-3). In vivo, both proteins also interact with ERp57 (4), a soluble luminal homologue of protein disulfide isomerase (PDI). Like PDI, ERp57 is composed of four thioredoxin-like domains with active site CXXC sequence motifs in the N-and C-terminal domains (5). During the folding of viral glycoproteins in the ER of living cells, ERp57 has been shown to form transient intermolecular disulfide bonds with glycoprotein substrates bound to CNX and CRT (6). When the association of CNX and CRT with glycoproteins is inhibited, the formation of intermolecular disulfide bonds with ERp57 is abrogated. Thus, the interaction between the glycoprotein substrates and either of the lectin chaperones seems to be required for the interaction with ERp57.The three-dimensional structure of both the CRT P-domain, CRT(189-288) (7) and the CNX ectodomain (including the CNX P-domain) (8) recently have been solved. They show that the P-domain comprises an unusual, extended hairpin fold, which in the crystal structure of the CNX ectodomain protrudes as a long arm from a compact, globular lectin domain. To gain insights into the cooperation of CRT and CNX with ERp57 during glycoprotein folding, we have characterized the interaction between the CRT P-domain and ERp57 by using biochemical methods and transverse relaxation-optimized spectroscopy (TROSY)-NMR. ...

In eukaryotic cells, the endoplasmic reticulum (ER) plays an essential role in the synthesis and maturation of a variety of important secretory and membrane proteins. For glycoproteins, the ER possesses a dedicated maturation system, which assists folding and ensures the quality of final products before ER release. Essential components of this system include the lectin chaperones calnexin (CNX) and calreticulin (CRT) and their associated co-chaperone ERp57, a glycoprotein specific thiol-disulfide oxidoreductase. The significance of this system is underscored by the fact that CNX and CRT interact with practically all glycoproteins investigated to date, and by the debilitating phenotypes revealed in knockout mice deficient in either gene. Compared to other important chaperone systems, such as the Hsp70s, Hsp90s and GroEL/GroES, the principles whereby this system works at the molecular level are relatively poorly understood. However, recent structural and biochemical data have provided important new insights into this chaperone system and present a solid basis for further mechanistic studies.

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 ...

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.Glyoxalase I and glyoxalase II are the two enzymes of the glyoxalase system. Glyoxalase I is an isomerase that catalyzes the formation of S-D-lactoylglutathione from the hemimercaptal adduct that forms nonenzymatically between glutathione and the 2-oxoaldehyde methylglyoxal (Fig. 1). Glyoxalase II then catalyzes the hydrolysis of the glutathione thiolester S-Dlactoylglutathione into reduced glutathione and D-lactate.

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