The structure in solution of the b domain of protein disulfide isomerase.

Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b and a. Both a and a domains contains an active site with the sequence motif -Cys-Gly-His-Cys-involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol. 7, 239-245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.

Identifying and characterizing a second structural domain of protein disulfide isomerase.

Recent protein engineering studies have confirmed the multidomain nature of protein disulfide isomerase previously suggested on the basis of analysis of its amino acid sequence. The boundaries of three domains, denoted a, a' and b, have been determined, and each domain has been expressed as an individual soluble folded protein. In this report, the boundaries of the final structural domain, b', are defined by a combination of restricted proteolysis and protein engineering approaches to complete our understanding of the domain organization of PDI. Using these data an optimized polypeptide construct has been prepared and characterized with a view to further structural and functional studies.

Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo.

Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast.

The functional properties of DsbG, a thiol-disulfide oxidoreductase from the periplasm of Escherichia coli.

Genetic studies have recently identified DsbG, a new member of the dsb group of redox proteins, which catalyze protein disulfide bond formation in the periplasm of Escherichia coli. We now demonstrate that DsbG functions primarily as an oxidant during protein disulfide bond formation, which is consistent with the low stability of its active site disulfide bond. There are indications, however, that the substrate range of DsbG may be narrower than the other periplasmic oxidative enzymes, DsbA and DsbC. Our observations further elaborate the pathway of disulfide bond formation in E. coli.

Enhanced catalysis of ribonuclease B folding by the interaction of calnexin or calreticulin with ERp57.

The endoplasmic reticulum is the site of folding, disulfide bond formation, and N-glycosylation of secretory proteins. Correctly folded proteins are exported from the endoplasmic reticulum, whereas incorrectly folded proteins are retained by a quality control system. The type I membrane-protein calnexin and its soluble homologue calreticulin are constituents of this system that recognize monoglucosylated N-linked glycans that are present on unfolded glycoproteins. Although several components of the quality control apparatus are well characterized, it is not known whether and how they interact with enzymes that catalyze protein folding. The endoplasmic reticulum protein ERp57 is homologous to protein-disulfide isomerase and can be cross-linked to the same monoglucosylated glycoproteins that bind to calnexin and calreticulin. The present study demonstrates that the disulfide isomerase activity of ERp57 on the refolding of monoglucosylated ribonuclease B is much greater when this glycoprotein is associated with calnexin or calreticulin. This result is in contrast to protein-disulfide isomerase, whose activity on monoglucosylated ribonuclease B is decreased in the presence of these lectins. No direct binding of monoglucosylated ribonuclease B or monoglucosylated glycans to ERp57 could be detected, but we show that ERp57 interacts directly with calnexin.

The multi-domain structure of protein disulfide isomerase is essential for high catalytic efficiency.

Protein disulfide isomerase (PDI) catalyzes protein folding linked to disulfide bond formation in secreted proteins. It consists of four major domains, denoted a, b, b' and a'. The a and a' domains each contain an active site motif, -CGHC-, which is directly involved in thiol-disulfide exchange reactions during catalysis. The roles of the b and b' domains and the functional necessity for the multi-domain structure of PDI are unknown. We now demonstrate that full catalytic activity requires the involvement of multiple PDI domains and that the b' domain has a particularly important role in catalysis. Reconstruction of the PDI molecule from the isolated a and a' domains results in a progressive increase in catalytic efficiency as further domains are added. These effects are especially significant in the catalysis of disulfide bond rearrangements in folded substrates, for which all the domains of the protein are required for maximum catalytic efficiency. It is likely that all of the domains of PDI participate in substrate binding interactions and that PDI has evolved its multidomain structure as an adaptation that allows it to catalyze transformations involving difficult conformational changes.

The b' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins.

Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core.

Contributions of substrate binding to the catalytic activity of DsbC.

DsbA and DsbC are involved in protein disulfide bond formation in the periplasm of Gram-negative bacteria. The two proteins are thought to fulfill different functions in vivo, DsbA as a catalyst of disulfide bond formation and DsbC as a catalyst of disulfide bond rearrangement. To explore the basis of this catalytic complementarity, the reaction mechanism of DsbC has been examined using unstructured model peptides that contain only one or two cysteine residues as substrates. The reactions between the various forms of the peptide and DsbC occur at rates up to 10(6)-fold faster than those that involve glutathione and DsbC, and they were constrained to occur at only one sulfur atom of disulfide bonds involving the peptide. Mixed disulfide complexes of DsbC and the peptide were 10(4)-fold more stable than the corresponding mixed disulfides with glutathione. These observations suggest that noncovalent binding interactions occur between the peptide and DsbC, which contribute to the very rapid kinetics of substrate utilization. The interactions between DsbC and the peptide appear to be more substantial than those between DsbA and the same peptide. The differences in the reaction of the peptide at the active sites of DsbA and DsbC provide insight into why DsbC is the better catalyst of disulfide bond rearrangement and how the active site chemistry of these structurally related proteins has been adapted to fulfill complementary functions.

The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules.

BACKGROUND: Protein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic reticulum, catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. Dissection of this protein into its individual domains has confirmed the presence of the a and a' domains, which are homologous to thioredoxin, having related structures and activities. The a and a' domains both contain a -Cys-Gly-His-Cys- active-site sequence motif. The remainder of the molecule consists primarily of two further domains, designated b and b' which are thought to be sequence repeats on the basis of a limited sequence similarity. The functions of the b and b' domains are unknown and, until now, the structure of neither domain was known. RESULTS: Heteronuclear nuclear magnetic resonance (NMR) methods have been used to determine the global fold of the PDI b domain. The protein has an alpha/beta fold with the order of the elements of secondary structure being beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-beta5+ ++-alpha4. The strands are all in a parallel arrangement with respect to each other, except for beta4 which is antiparallel. The arrangement of the secondary structure elements of the b domain is identical to that found in the a domain of PDI and in the ubiquitous redox protein thioredoxin; the three-dimensional folding topology of the b domain is also very similar to that of these proteins. CONCLUSIONS: Our determination of the global fold of the b domain of PDI by NMR reveals that, like the a domain, the b domain contains the thioredoxin motif, even though the b domain has no significant amino-acid sequence similarities to any members of the thioredoxin family. This observation, together with indications that the b' domain adopts a similar fold, suggests that PDI consists of active and inactive thioredoxin modules. These modules may have been adapted during evolution to provide PDI with its complete spectrum of enzymatic activities.

Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

Proteins with the thioredoxin fold have widely differing stabilities of the disulfide bond that can be formed between the two cysteines at their active site sequence motif Cys1-Xaa2-Yaa3-Cys4. This is believed to be regulated not by varying the disulfide bond itself, but by modulating the stability of the dithiol form of the protein through interactions with the ionized form of the Cys1 thiol group. A consistent relationship between disulfide bond stability and Cys1 thiol pKa value is found here for DsbA, thioredoxin, and the N-terminal thioredoxin-like domain of protein disulfide isomerase (PDI a), which has a very low thiol pKa value of 4.5. This thiolate anion is stabilized by 5.7 kcal/mol in the dithiol form, giving rise to the corresponding instability of the disulfide bond and the oxidizing properties of PDI a. Electrostatic interactions in the active site of the PDI a-domain have been characterized in order to understand the physical basis of this stabilization. Linkage with the ionization of the imidazole group of His3 in the active site demonstrates that this charge-charge interaction contributes 1.1 kcal/mol. The remainder of the stabilization is believed to be due primarily to interactions with the partial positive charges at the N-terminus of an alpha-helix, which are exceedingly sensitive to charges of surrounding residues.

Identifying and characterizing a structural domain of protein disulfide isomerase.

Protein disulfide isomerase (PDI) appears on the basis of its primary structure to be a multidomain protein, but the number and nature of the domains has been uncertain. Two of the domains, a and a', which are homologous to thioredoxin and active in catalysis of disulfide bond formation, have been identified and characterized previously. Sections of the N-terminal half of the PDI sequence have been expressed and the limits of their folded structures delineated by limited proteolysis. In addition to the a-domain, the boundaries of a domain with no activity on thiol/disulfide groups, designated b, have been identified. This domain has been produced independently; its cooperative unfolding transition and its CD and NMR spectra confirm that it is an autonomously folded structure in isolation and when part of PDI. Fusion of the b-domain to the a-domain, as occurs naturally in the first half of PDI, did not alter substantially the catalytic activity of the a-domain. It still catalyzes only a subset of the thiol/disulfide exchange reactions of intact PDI and has a reduced ability to catalyze protein disulfide rearrangements. The a- and b-domains account structurally for virtually all of the first half of the PDI polypeptide chain, and it is very unlikely that there exists a proposed third domain homologous to the estrogen receptor. The b-domain exhibits some sequence homology to calsequestrin, a calcium binding protein from the sarcoplasmic reticulum of muscle.

Structure determination of the N-terminal thioredoxin-like domain of protein disulfide isomerase using multidimensional heteronuclear 13C/15N NMR spectroscopy.

As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the structure of a fragment corresponding to the first 120 residues of its sequence has been determined using heteronuclear multidimensional NMR techniques. As expected from its primary structure homology, the fragment has the thioredoxin fold. Similarities and differences in their structures help to explain why thioredoxins are reductants, whereas PDI is an oxidant of protein thiol groups. The results confirm that PDI has a modular, multidomain structure, which will facilitate its structural and functional characterization.

The roles of partly folded intermediates in protein folding.

Proteins can fold very rapidly, undoubtedly because they do not do so simply by random searching. The stable, partly folded species that can be detected during protein refolding are, however, of uncertain kinetic significance. The available kinetic evidence indicates that the intermediates that are most responsible for the rapidity of folding are extremely unstable and not populated detectably; they are less extreme versions of the transition state for folding. Protein folding is most readily studied when it is coupled to disulfide formation, which has the advantages that the intermediates can be characterized in detail and their kinetic roles determined unambiguously. The most important aspects of the disulfide folding pathway of BPTI are understood to at least a first approximation, and several other protein disulfide folding pathways are known in outline. These pathways demonstrate that disulfide folding is not intrinsically different from that not involving disulfide formation. Partly folded conformations can increase the rate of folding somewhat by causing productive disulfide bonds to be populated preferentially, but the most important folding intermediates are not detectable. The essence of folding is to build up the cooperativity between the individual interactions that is necessary for a stable conformation.

Characterization of the active site cysteine residues of the thioredoxin-like domains of protein disulfide isomerase.

The dithiol/disulfide active sites of each of the two isolated thioredoxin-like domains of protein disulfide isomerase (PDI) expressed in Escherichia coli have been characterized in order to understand their catalytic mechanisms and their functions in PDI. In each of the folded domains, as in other proteins of the thioredoxin family, only one of the cysteine residues of the active site sequence -Cys-Gly-His-Cys- is accessible, and its thiol group is highly reactive and has a low pKa value. The kinetics and equilibria have been measured of the reactions between the active site cysteine residues and glutathione, the predominant thiol/disulfide reagent of the endoplasmic reticulum. A disulfide bond can be formed very rapidly between the pair of cysteine residues of each domain, but each disulfide bond is very unstable and reacts rapidly with reduced glutathione. The very low stabilities of these disulfide bonds, which destabilize the protein structures, account for the efficiency with which PDI and each of the isolated domains can introduce disulfide bonds into proteins. These kinetics and equilibrium data go far in helping to understand the catalytic mechanism of PDI and its individual domains.

Nuclear magnetic resonance characterization of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

A genetically engineered protein consisting of the 120 residues at the N-terminus of human protein disulfide isomerase (PDI) has been characterized by 1H, 13C, and 15N NMR methods. The sequence of this protein is 35% identical to Escherichia coli thioredoxin, and it has been found also to have similar patterns of secondary structure and beta-sheet topology. The results confirm that PDI is a modular, multidomain protein. The last 20 residues of the N-terminal domain of PDI are some of those that are similar to part of the estrogen receptor, yet they appear to be an intrinsic part of the thioredoxin fold. This observation makes it unlikely that any of the segments of PDI with similarities to the estrogen receptor comprise individual domains.

Functional properties of the individual thioredoxin-like domains of protein disulfide isomerase.

The two thioredoxin-like domains of human protein disulfide isomerase (PDI) have been produced in bacteria as individual soluble, folded protein molecules, and their functional properties have been compared to those of intact PDI. The two individual domains were very similar in their functional properties, and there were no indications of synergy between them, so it is unlikely that they have intrinsically different functions in PDI. Both domains efficiently introduced disulfide bonds into unfolded model proteins and peptides but were less efficient than PDI with folded substrate protein molecules. Relative to PDI, neither domain had substantial activity in catalyzing disulfide bond isomerization. This pattern of activities is very similar to that of the bacterial catalyst DsbA and probably reflects similarities in the catalytic mechanisms of these proteins. The differences in activity between PDI and its thioredoxin-like domains suggest that other features of the PDI molecule are also required for its complete range of thiol-disulfide exchange activities.

Refolding of bovine pancreatic trypsin inhibitor via non-native disulphide intermediates.

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species. The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30-51, 5-14) and (30-51, 5-38). Moreover, the rates of folding through each of these intermediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathway, and their roles are readily explicable on the basis of their conformations.

Catalytic mechanism of DsbA and its comparison with that of protein disulfide isomerase.

The mechanism of action of the bacterial periplasm protein DsbA in introducing disulfide bonds into proteins was studied by its action on a model disordered peptide containing only two cysteine residues. Most of the reactions between the various thiol and disulfide forms of the peptide and of DsbA could be measured directly. All those involving DsbA occurred 10(2)-10(6) times more rapidly than is normally observed between other typical thiols and disulfides; DsbA apparently stabilizes the transition state of thiol-disulfide exchange. The reactions between DsbA and the peptide were even more rapid, and they were constrained to occur at only one sulfur atom of disulfide bonds involving the peptide. Both observations indicate that noncovalent binding interactions also occur between DsbA and the peptide, and the expected effect of binding between reactants on rates of reaction was quantified. Small quantities of DsbA had catalytic effects on the reaction between the peptide and glutathione, similar to those observed previously with the eukaryotic catalyst protein disulfide isomerase. The known reactions of DsbA could account quantitatively for these effects and indicated that the apparent catalysis was the result of the separate and sequential rapid reactions of the peptide and of glutathione at the active site of DsbA. DsbA did not catalyze the conformational changes involved in forming an intramolecular disulfide bond in the peptide; its catalytic effects were simply due to its rapid participation in thiol-disulfide exchange reactions. Protein disulfide isomerase is likely to function very similarly to DsbA.

Mechanisms and catalysts of disulfide bond formation in proteins.

The formation of disulphide bonds is an important co- and post-translational event in the biosynthesis of many extracellular proteins that is often coupled to protein folding. Progress in understanding how disulphide bonds form in model proteins provides insight into how the process can be manipulated to optimize the production of engineered proteins.