ERp57 and PDI: multifunctional protein disulfide isomerases with similar domain architectures but differing substrate-partner associations.

Secretory proteins become folded and acquire stabilizing disulfide bonds in the endoplasmic reticulum (ER). Correct disulfide bond formation is a key step in ER quality control (ERQC). Proteins with incorrect disulfide bonds are recognized by the quality control machinery and are retrotranslocated into the cytosol where they are degraded by the proteasome. The mammalian ER contains 17 disulfide isomerases and at least one of them, ERp57, works in conjunction with the ER lectin-like chaperones calnexin and calreticulin. The targeting of ERp57 to calnexin-calreticulin is mediated by its noncatalytic b' domain, and analogous domains in other disulfide isomerases likely determine their substrate and partner preferences. This review discusses some explanations for the multiplicity of disulfide isomerases and highlights structural differences in the b' domains of PDI and ERp57 as an example of how noncatalytic domains define specialized roles in oxidative folding.     

Substrate Specificity of the Oxidoreductase ERp57 Is Determined Primarily by Its Interaction with Calnexin and Calreticulin

The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins.The ability to form disulfide bonds within proteins entering the secretory pathway is essential for cell survival and occurs within the endoplasmic reticulum (ER).³ For proteins with few disulfides, the process can be catalyzed by oxidation of cysteine residues to form the correct, native disulfide; however, for proteins with several disulfides, an isomerization reaction is also required to correct non-native disulfides formed following oxidation (1). Both these reactions are catalyzed by a group of ER-resident proteins that belong to the protein disulfide isomerase (PDI) family, which comprises over 17 members (2). It is well established that PDI and several other family members are able to catalyze the formation and isomerization of disulfides in vitro, although the exact function of each of the family members in vivo is unknown. It is still an open question as to whether they all catalyze similar reactions and have distinct substrate specificities or whether they have distinct enzymatic functions related to the breaking and formation of disulfides.For one member of the PDI family, the function and substrate specificity is a little clearer. ERp57 has been shown previously to interact specifically with glycoproteins during their folding (3). The enzyme is physically associated with either calnexin or calreticulin (4) and is therefore ideally placed to catalyze correct disulfide formation within proteins entering the calnexin/calreticulin cycle (referred to subsequently just as the calnexin cycle). In addition, the ability of ERp57 to catalyze the refolding of substrates in vitro is greatly enhanced if the substrate is bound to calnexin (5). Recently, substrates for the reduction or isomerization reaction catalyzed by ERp57 have been identified by trapping mixed disulfides between enzyme and substrate (6). Strikingly, there was an overrepresentation of substrate proteins with cysteine-rich domains containing little secondary structure, suggesting that the main function of ERp57 is in the isomerization of non-native disulfides. ERp57 has also been shown to function independently from the calnexin cycle. It is a component of the MHC class I loading complex where it forms a disulfide-linked complex with tapasin and is thought to either stabilize the complex or facilitate correct assembly of class I molecules (7, 8). Recently, ERp57 has been demonstrated to isomerize interchain disulfides in the major capsid protein, VP1, of simian virus 40 (9). The ability to dissociate VP1 pentamers by ERp57 does not require the substrate to interact with the calnexin cycle. Hence, it is still unclear how ERp57 recognizes its substrates, and in particular, whether this recognition is solely determined by an interaction with the calnexin cycle.The recognition of substrates by PDI is somewhat clearer in that one particular domain within the protein (the b′ domain) has been shown to be primarily responsible for substrate recognition and peptide binding (10). The corresponding domain within ERp57 has been shown to be responsible for interaction with the calnexin cycle (11), suggesting that for ERp57, substrate recognition must occur outside this domain or is determined solely by substrate interaction with calnexin via its oligosaccharide side chain. Hence, the aim of our study was to evaluate the necessity of the calnexin cycle both for ERp57 to recognize its substrates and for correct folding of glycoproteins. ERp57 was found to be required for the efficient folding of one substrate, influenza virus hemagglutinin (HA), but only when it entered the calnexin cycle. HA did not require ERp57 to fold if it was blocked from entering the calnexin cycle. In contrast, β1-integrin does not fold efficiently either if ERp57 was depleted or if ERp57 is blocked from entering the calnexin cycle (6). Although ERp57 may be dispensable for the folding of some glycoproteins, the interaction with calnexin commits them to an ERp57-dependent fate. We also found that the majority of ERp57 substrates need to enter the calnexin cycle to be acted upon by the enzyme, demonstrating that substrate specificity is primarily dependent upon substrate entry into the calnexin cycle.     

Substrate-specific requirements for UGT1-dependent release from calnexin

Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER.     

ERp57 Functions as a Subunit of Specific Complexes Formed with the ER Lectins Calreticulin and Calnexin

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.     

Crystal structure of the bb' domains of the protein disulfide isomerase ERp57

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.     

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein

Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease.     

Endoplasmic reticulum chaperones are involved in the morphogenesis of rotavirus infectious particles

The final assembly of rotavirus particles takes place in the endoplasmic reticulum (ER). In this work, we evaluated by RNA interference the relevance to rotavirus assembly and infectivity of grp78, protein disulfide isomerase (PDI), grp94, calnexin, calreticulin, and ERp57, members of the two ER folding systems described herein. Silencing the expression of grp94 and Erp57 had no effect on rotavirus infectivity, while knocking down the expression of any of the other four chaperons caused a reduction in the yield of infectious virus of about 50%. In grp78-silenced cells, the maturation of the oligosaccharide chains of NSP4 was retarded. In cells with reduced levels of calnexin, the oxidative folding of VP7 was impaired and the trimming of NSP4 was accelerated, and in calreticulin-silenced cells, the formation of disulfide bonds of VP7 was also accelerated. The knockdown of PDI impaired the formation and/or rearrangement of the VP7 disulfide bonds. All these conditions also affected the correct assembly of virus particles, since compared with virions from control cells, they showed an altered susceptibility to EGTA and heat treatments, a decreased specific infectivity, and a diminished reactivity to VP7 with monoclonal antibody M60, which recognizes only this protein when its disulfide bonds have been correctly formed. In the case of grp78-silenced cells, the virus produced bound less efficiently to MA104 cells than virus obtained from control cells. All these results suggest that these chaperones are involved in the quality control of rotavirus morphogenesis. The complexity of the steps of rotavirus assembly that occur in the ER provide a useful model for studying the organization and operation of the complex network of chaperones involved in maintaining the quality control of this organelle.     

Independent and cooperative roles of N-glycans and molecular chaperones in the folding and disulfide bond formation of the low-density lipoprotein (LDL) receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large receptor that contains extensive glycosylation sites and disulfide bonds. Here we analyzed how N-linked glycosylation and molecular chaperones function during LRP folding. Treatment of cells with a glycosylation inhibitor tunicamycin significantly impaired LRP folding, although binding to receptor-associated protein (RAP), a specialized chaperone for LRP, was not affected. The effects of tunicamycin on LRP folding were not due to an inhibition of RAP glycosylation since a mutant RAP that harbors a mutation at its sole glycosylation site was still capable of promoting LRP folding. The roles of N-linked glycosylation and the lectin chaperone, calnexin, in LRP folding were further dissected using LRP minireceptors that carry mutations at individual glycosylation sites. Interestingly, we found that RAP interacts with oxidoreductase ERp57 and mediates its interaction with LRP. Since previous studies have shown that N-glycan-bound calnexin/calreticulin are also capable of recruiting ERp57, our results suggest that N-linked glycosylation and RAP can independently and cooperatively recruit oxidoreductases to facilitate protein folding and proper disulfide bond formation.     

Calnexin,calreticulin, and ERp57 cooperate in disulfide bond formation in human CD1d heavy chain

Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and beta(2)-microglobulin (beta(2)m) in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need beta(2)m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with beta(2)m. These differences led us to investigate early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. Here we show that CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin.     

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.     

Functions of ERp57 in the folding and assembly of major histocompatibility complex class I molecules

ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain alpha(3) domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-beta(2)-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and beta(2) -microglobulin does not depend on an oxidized alpha(3) domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process.     

Mixed-disulfide folding intermediates between thyroglobulin and endoplasmic reticulum resident oxidoreductases ERp57 and protein disulfide isomerase

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.     

Two phases of disulfide bond formation have differing requirements for oxygen

Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for oxygen in protein folding. Here we demonstrate the existence of two phases of disulfide bond formation in living mammalian cells, with differential requirements for oxygen. Disulfide bonds introduced rapidly during protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent. Other protein maturation processes in the secretory pathway, including ER-localized N-linked glycosylation, glycan trimming, Golgi-localized complex glycosylation, and protein transport, occur independently of oxygen availability. These results suggest that an alternative electron acceptor is available transiently during an initial phase of disulfide bond formation and that post-translational oxygen-dependent disulfide bond formation causes hypoxia-induced ER stress.     

Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system

Calnexin and ERp57 act cooperatively to ensure a proper folding of proteins in the endoplasmic reticulum (ER). Calnexin contains two domains: a lectin domain and an extended arm termed the P-domain. ERp57 is a protein disulfide isomerase composed of four thioredoxin-like repeats and a short basic C-terminal tail. Here we show direct interactions between the tip of the calnexin P-domain and the ERp57 basic C-terminus by using NMR and a novel membrane yeast two-hybrid system (MYTHS) for mapping protein interactions of ER proteins. Our results prove that a small peptide derived from the P-domain is active in binding ERp57, and we determine the structure of the bound conformation of the P-domain peptide. The experimental strategy of using the MYTHS two-hybrid system to map interaction sites between ER proteins, together with NMR, provides a powerful new strategy for establishing the function of ER complexes.     

Overexpression of human calnexin in yeast improves measles surface glycoprotein solubility

The limitations of high-level expression of virus surface proteins in yeast are not well understood. The inefficiency of yeast to produce active human virus surface glycoproteins, as well as other mammalian glycoproteins, is usually explained by the inefficient folding of the glycoprotein into its characteristic and functional three-dimensional structure from a random coil. The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. To improve folding and solubility of viral surface glycoprotein, the genes encoding human ER resident chaperones calnexin, calreticulin, immunoglobin binding protein (BiP), protein disulfide isomerase (PDI) and foldase (ERp57) were coexpressed together with hemagglutinin gene from measles virus in the yeast Saccharomyces cerevisiae. The effect of coexpressing chaperones on the total yield of measles virus hemagglutinin (MeH) as well as the intracellular fate of the glycoprotein was determined. Our results demonstrated that coexpression of human calnexin noticeably enhanced the quantity of the soluble glycosylated form of MeH in yeast. The coexpression of human calreticulin-, PDI-, ERp57- and BiP-encoding genes did not improve the quality of recombinant MeH.     

Influence of the oxidoreductase ER57 on the folding of an antibody fab fragment

Oxidation and folding of secretory proteins in the endoplasmic reticulum (ER) depends on the presence of chaperones and oxidoreductases. Two of the oxidoreductases present in the ER of mammalian cells are protein disulfide isomerase (PDI) and ERp57. In this study, we investigated the influence of ERp57 on the in vitro reoxidation and refolding of an antibody Fab fragment. Our results show that ERp57 shares functional properties with PDI and that both are clearly different from other oxidoreductases. The reactivation of the denatured and reduced Fab fragment was enhanced significantly in the presence of ERp57 with kinetics and redox dependence of the reactivation reaction comparable to those obtained for PDI. These properties were not influenced by the presence of calnexin. Furthermore, whereas PDI cooperates with the immunoglobulin heavy chain binding protein (BiP), no synergistic effect could be observed for BiP and ERp57. These results indicate that the cooperation of the two oxidoreductases with different partner proteins may explain their different roles in the folding of proteins in the ER.     

Functional Relationship between Protein Disulfide Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins

To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.     

NMR structures of 36 and 73-residue fragments of the calreticulin P-domain

Calreticulin (CRT) is an abundant, soluble molecular chaperone of the endoplasmic reticulum. Similar to its membrane-bound homolog calnexin (CNX), it is a lectin that promotes the folding of proteins carrying N-linked glycans. Both proteins cooperate with an associated co-chaperone, the thiol-disulfide oxidoreductase ERp57. This enzyme catalyzes the formation of disulfide bonds in CNX and CRT-bound glycoprotein substrates. Previously, we solved the NMR structure of the central proline-rich P-domain of CRT comprising residues 189-288. This structure shows an extended hairpin topology, with three short anti-parallel beta-sheets, three small hydrophobic clusters, and one helical turn at the tip of the hairpin. We further demonstrated that the residues 225-251 at the tip of the CRT P-domain are involved in direct contacts with ERp57. Here, we show that the CRT P-domain fragment CRT(221-256) constitutes an autonomous folding unit, and has a structure highly similar to that of the corresponding region in CRT(189-288). Of the 36 residues present in CRT(221-256), 32 form a well-structured core, making this fragment one of the smallest known natural sequences to form a stable non-helical fold in the absence of disulfide bonds or tightly bound metal ions. CRT(221-256) comprises all the residues of the intact P-domain that were shown to interact with ERp57. Isothermal titration microcalorimetry (ITC) now showed affinity of this fragment for ERp57 similar to that of the intact P-domain, demonstrating that CRT(221-256) may be used as a low molecular mass mimic of CRT for further investigations of the interaction with ERp57. We also solved the NMR structure of the 73-residue fragment CRT(189-261), in which the tip of the hairpin and the first beta-sheet are well structured, but the residues 189-213 are disordered, presumably due to lack of stabilizing interactions across the hairpin.