Disulfide-bonded Intermediates Research Articles

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 Structure of a Two-Disulfide Intermediate Assists in Elucidating the Oxidative Folding Pathway of a Cyclic Cystine Knot Protein

We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.

Development of a novel method to populate native disulfide-bonded intermediates for structural characterization of proteins: implications for the mechanism of oxidative folding of RNase A.

RNase A, a model protein for oxidative folding studies, has four native disulfide bonds. The roles of des [40-95] and des [65-72], the two native-like structured three-disulfide-bonded intermediates populated between 8 and 25 degrees C during the oxidative folding of RNase A, are well characterized. Recent work focuses on both the formation of these structured disulfide intermediates from their unstructured precursors and on the subsequent oxidation of the structured species to form the native protein. The major obstacles in this work are the very low concentration of the precursor species and the difficulty of isolating some of the structured intermediates. Here, we demonstrate a novel method that enables the native disulfide-bonded intermediates to be populated and studied regardless of whether they have stable structure and/or are present at low concentrations during the oxidative folding or reductive unfolding process. The application of this method enabled us to populate and, in turn, study the key intermediates with two native disulfide bonds on the oxidative folding pathway of RNase A; it also facilitated the isolation of des [58-110] and des [26-84], the other two native-like structured des species whose isolation had thus far not been possible.

PII: S0969-2126(01)00700-6

PII: S0969-2126(01)00700-6

Slow Folding of Three-Fingered Toxins Is Associated with the Accumulation of Native Disulfide-Bonded Intermediates

Snake neurotoxins are short all-beta proteins that display a complex organization of the disulfide bonds: two bonds connect consecutive cysteine residues (C43-C54, C55-C60), and two bonds intersect when bridging (C3-C24, C17-C41) to form a particular structure classified as "disulfide beta-cross". We investigated the oxidative folding of a neurotoxin variant, named alpha62, to define the chemical nature of the three-disulfide intermediates that accumulate during the process in order to describe in detail its folding pathway. These folding intermediates were separated by reverse-phase HPLC, and their disulfide bonds were identified using a combination of tryptic hydrolysis, manual Edman degradation, and mass spectrometry. Two dominant intermediates containing three native disulfide bonds were identified, lacking the C43-C54 and C17-C41 pairing and therefore named des-[43-54] and des-[17-41], respectively. Both species were individually allowed to reoxidize under folding conditions, showing that des-[17-41] was a fast-forming nonproductive intermediate that had to interconvert into the des-[43-54] isomer before forming the native protein. Conversely, the des-[43-54] intermediate appeared to be the immediate precursor of the oxidized neurotoxin. A kinetic model for the folding of neurotoxin alpha62 which fits with the observed time-course accumulation of des-[17-41] and des-[43-54] is proposed. The effect of turn 2, located between residues 17 and 24, on the overall kinetics is discussed in view of this model.

Mutational analysis of hydrogen bonding residues in the BPTI folding pathway

Nine BPTI variants with replacements that remove one or more hydrogen bonds from the native protein were constructed, and the folding pathways of these proteins were determined by isolating and identifying the disulfide-bonded intermediates that accumulated during unfolding and refolding. The forward and reverse rate constants for the individual steps in the folding pathways for each protein were measured, providing a detailed description of the energetic effects of the substitutions. The native forms of eight of the nine variants were measurably destabilized, by 1–7 kcal/mol (1 cal = 4.184 J), with an average effect of 1.6 kcal/mol per hydrogen bond removed. The folding pathways for the variants were found to be similar to that previously described for the wild-type protein, with the kinetically preferred mechanism involving intramolecular rearrangements of intermediates with two disulfide bonds. Some of the substitutions, however, significantly destabilized the major intermediates and broadened the distribution of species with one or two disulfide bonds, thus identifying residues that play important roles in stabilizing the normal intermediates and defining specificity in the folding process. The kinetic data also suggest that one residue, Asn43, may play a distinctive role in defining the BPTI folding mechanism. Replacement of this residue with either Gly or Ala appeared to stabilize the major transition states for folding and unfolding. In the native protein, the side-chain of Asn43 participates directly in the hydrogen bonding pattern of the central β-sheet, and the kinetic behavior of the Asn43 variants suggests that the major energy barriers in folding and unfolding may be due in part to the steric constraints imposed by this structural element, together with those imposed by the chemical transition states for thiol-disulfide exchange.

ER60/ERp57 forms disulfide-bonded intermediates with MHC class I heavy chain.

SPECIFIC AIMSIn this study, we investigated the role of ER60 (ERp57/GRP58) in the formation of disulfide bonds within the major histocompatibility (MHC) class I heavy chain. We also examined the role of calnexin in recruiting ER60 into early folding complexes with the nascent class I heavy chain (HC).PRINCIPAL FINDINGS1. ER60/ERp57 forms disulfide-bonded intermediates with class I heavy chainMouse EL4 cells were metabolically labeled for 15 min with [35S] cysteine/methionine. After labeling, the cells were washed in PBS containing N-ethyl-maleimide (NEM), a membrane-permeable alkylating agent, and lysed in 1% digitonin lysis buffer containing NEM to trap unreacted cysteines. Protein complexes containing ER60 were isolated by immunoprecipitation using anti-ER60 sera. To identify a disulfide-bonded intermediate, the complexes were resolved on a 2-dimensional gel system using nonreducing gels in the first dimension. Proteins were resolved on 8% polyacrylamide gels in capillary tubes. The gels were then extru...

Early events in the disulfide-coupled folding of BPTI.

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.

Regeneration of bovine pancreatic ribonuclease A: detailed kinetic analysis of two independent folding pathways.

The regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol at 25 degrees C, pH 8.0, proceeds through two separate pathways in which separate nativelike three-disulfide species are populated. The populations of these two three-disulfide species during the regeneration process have been monitored directly through the use of a reduction pulse. A detailed kinetic analysis of the regeneration process using improved experimental procedures and data analysis has been carried out to obtain rate constants for disulfide interconversion among the various disulfide-bonded intermediates. This analysis indicates that these two pathways can account for essentially 100% of the native protein regenerated and that the relative amount of native protein regenerated through these two pathways is insensitive to the redox conditions used. These results indicate that the rate-determining step in both pathways involves formation of the nativelike three-disulfide species, a step in which most of the conformational folding takes place. The experimentally determined rate constants indicate that these two pathways are sufficient to explain the differences in the temperature dependence of the regeneration rate with different redox reagents. In addition, the population of a fully oxidized species that contains three native disulfide bonded pairs and a dithiothreitol bridging cysteines 65 and 72 has been observed.

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide-bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide-bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild-type and mutant proteins.

The pro region of BPTI facilitates folding

The in vitro folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been described previously in terms of the disulfide-bonded intermediates that accumulate during folding of the protein. Folding is slow, occurring in hours at pH 7.3, 25°C. In addition, approximately half of the BPTI molecules become trapped as a dead-end, native-like intermediate. In vivo, BPTI is synthesized as a precursor protein that includes a 13 residue amino-terminal pro region. This pro region contains a cysteine residue. We find that, in vitro, both the rate of formation and the yield of properly folded BPTI are increased substantially in a recombinant model of pro-BPTI. The cysteine residue is necessary for this effect. Moreover, a single cysteine residue, tethered to the carboxy-terminal end of BPTI with a flexible linker of repeating Ser-Gly-Gly residues, is sufficient to assist in disulfide formation. Thus, the pro region appears to facilitate folding by providing a tethered, solvent-accessible, intramolecular thiol-disulfide reagent.

Native and non-native intermediates in the BPTI folding pathway.

Recent studies of the disulfide-bonded intermediates in the refolding of bovine pancreatic trypsin inhibitor (BPTI) indicate that the most stable intermediates can take on much or all of the structure of the fully folded protein. Native-like structure in the intermediates probably causes steric inhibition of direct sequential formation of the three disulfides found in the native protein, thus accounting for the role of intramolecular rearrangements in the folding mechanism of this small protein.

Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum.

By resolving immunoprecipitates on nonreducing sodium dodecyl sulfate gels, we have detected several disulfide-bonded intermediates in folding within the endoplasmic reticulum of newly made H1 subunits of the asialoglycoprotein receptor. H1 in the endoplasmic reticulum (ER) can be partially unfolded by treatment of cells with dithiothreitol, but H1 in Golgi or post-Golgi organelles is resistant to such unfolding. This defines a late step in H1 folding that occurs just prior to exit from the ER. Depletion of calcium from the endoplasmic reticulum, either by treatment with A23187 or thapsigargin, has no effect on folding or secretion of newly made albumin, but totally blocks H1 maturation from the ER. No ER intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in H1 folding requires a high Ca2+ concentration in the ER lumen. As judged by cross-linking experiments, formation of H1 dimers and trimers occurs immediately after biosynthesis of the peptide chain, before monomer folding, and occurs normally in cells in which ER Ca2+ is reduced and where the monomer never folds properly. Calcium is essential for the asialoglycoprotein receptor to bind galactose, and our results suggest that Ca2+ is also essential for the receptor polypeptides to fold in the ER.