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• W2092341922 abstract "Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bonded proteins and also a multifunctional polypeptide that acts as the β-subunit in the prolyl 4-hydroxylase α2β2-tetramer (P4H) and the microsomal triglyceride transfer protein αβ-dimer. The principal peptide-binding site of PDI is located in the b′ domain, but all domains contribute to the binding of misfolded proteins. Mutations in the C-terminal part of the a′ domain have significant effects on the assembly of the P4H tetramer and other functions of PDI. In this study we have addressed the question of whether these mutations in the C-terminal part of the a′ domain, which affect P4H assembly, also affect peptide binding to PDI. We observed a strong correlation between P4H assembly competence and peptide binding; mutants of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivity in these assays and indicators of conformational disruption, such as protease sensitivity. Peptide binding activity could be restored in inactive, protease-sensitive mutants by selective proteolytic removal of the mutated a′ domain. Hence we propose that structural changes in the a′ domain indirectly affect peptide binding to the b′ domain. Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bonded proteins and also a multifunctional polypeptide that acts as the β-subunit in the prolyl 4-hydroxylase α2β2-tetramer (P4H) and the microsomal triglyceride transfer protein αβ-dimer. The principal peptide-binding site of PDI is located in the b′ domain, but all domains contribute to the binding of misfolded proteins. Mutations in the C-terminal part of the a′ domain have significant effects on the assembly of the P4H tetramer and other functions of PDI. In this study we have addressed the question of whether these mutations in the C-terminal part of the a′ domain, which affect P4H assembly, also affect peptide binding to PDI. We observed a strong correlation between P4H assembly competence and peptide binding; mutants of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivity in these assays and indicators of conformational disruption, such as protease sensitivity. Peptide binding activity could be restored in inactive, protease-sensitive mutants by selective proteolytic removal of the mutated a′ domain. Hence we propose that structural changes in the a′ domain indirectly affect peptide binding to the b′ domain. protein-disulfide isomerase disuccinimidyl glutarate prolyl 4-hydroxylase microsomal triglyceride transfer protein polyvinylidene difluoride Protein-disulfide isomerase (PDI),1 an abundant protein of the eukaryotic endoplasmic reticulum, is a catalyst of disulfide bond formation and rearrangement in the course of protein folding (for review see Ref. 1.Freedman R.B. Klappa P. Bukau B. Molecular Chaperones and Protein Folding. Harwood Academic Press, London1999: 437-459Google Scholar). A molecular interpretation of PDI activity is made difficult by the fact that PDI is multi-functional in the cell. In addition, it has a wide range of actions in vitro and consists of multiple domains. Furthermore, there is as yet no high resolution structure of full-length PDI. Current efforts are focused on analyzing the domain structure of PDI and in establishing the roles of specific domains in specific functional activities.It is now clear (2.Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 3.Freedman R.B. Gane P.J. Hawkins H.C. Hlodan R. McLaughlin S.H. Parry J.W. Biol. Chem. Hoppe-Seyler. 1998; 379: 321-328Crossref PubMed Scopus (51) Google Scholar) that PDI has a structural organization based on duplicated sequence modules (Fig. 1). The full-length protein is constructed of four structural domains with homologous thioredoxin folds plus a C-terminal acidic extension. The homologous a and a′ sequence modules contain the active site motif -WCGHC- and show significant sequence identity to thioredoxin; a high resolution NMR analysis of the recombinanta domain confirms that it has the thioredoxin fold. The homologous b and b′ modules do not show significant sequence similarity to the a domain, but NMR analysis has revealed that the b domain also exhibits the thioredoxin fold (2.Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 4.Kemmink J. Dijkstra K. Mariani M. Scheek R.M. Penka E. Nilges M. Darby N.J. J. Biomol. NMR. 1999; 13: 357-368Crossref PubMed Scopus (68) Google Scholar). Analysis of the properties of individual domains as catalysts of simple thiol:disulfide oxidoreduction and of more complex protein folding linked to disulfide isomerization shows that the a and a′ domains function effectively as simple thiol:disulfide oxidoreductases but that the remaining domains are required for full activity in catalyzing protein folding associated with the formation of native disulfide bonds (5.Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). No specific function has yet been ascribed to the b domain, but cross-linking studies demonstrate that the b′ domain provides the principal peptide-binding site (6.Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (292) Google Scholar).In addition to its role in catalyzing protein folding, PDI has also been described as the β-subunit of two hetero-oligomeric proteins, namely prolyl-4-hydroxylase (P4H) (7.Pihlajaniemi T. Helaakoski T. Tasanen K. Myllyla R. Huhtala M.L. Koivu J. Kivirikko K.I. EMBO J. 1987; 6: 643-649Crossref PubMed Scopus (330) Google Scholar) and the microsomal triglyceride transferase complex (MTP) (8.Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9801-9807Abstract Full Text PDF Google Scholar, 9.Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Biochemistry. 1991; 30: 9728-9735Crossref PubMed Scopus (154) Google Scholar). MTP is obligatory for the assembly of apoB-containing lipoproteins, whereas P4H is important in the post-translational formation of 4-hydroxyproline in collagen in the endoplasmic reticulum (10.Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (235) Google Scholar, 11.Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). It appears, both from attempts at reassociating these complexes after dissociation in vitro(11.Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar) and from studies on assembly of the complexes at biosynthesis, that PDI is required to prevent the aggregation of its partner subunits during either initial folding in the cell or refolding in vitro (12.Vuori K. Pihlajaniemi T. Myllyla R. Kivirikko K.I. EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (127) Google Scholar, 13.Vuori K. Pihlajaniemi T. Marttila M. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7467-7470Crossref PubMed Scopus (114) Google Scholar, 14.John D.C. Grant M.E. Bulleid N.J. EMBO J. 1993; 12: 1587-1595Crossref PubMed Scopus (94) Google Scholar, 15.Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar). Site-directed mutagenesis of the β-subunit/PDI demonstrated that the active site cysteine residues of PDI were not essential for the assembly and activity of active P4H tetramer or of MTP (12.Vuori K. Pihlajaniemi T. Myllyla R. Kivirikko K.I. EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (127) Google Scholar, 15.Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar).The question then arises as to which domains or regions of PDI are involved in or required for its interaction with the other subunits in these hetero-oligomeric complexes. Analysis of deletion and point mutants of PDI recently demonstrated that the acidic C-terminal extension is not critical for the disulfide isomerase activity of PDI or for its ability to assemble into active P4H (16.Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helaakoski T. Notbohm H. Kivirikko K.I. EMBO J. 1999; 18: 65-74Crossref PubMed Scopus (53) Google Scholar). However, deletion and point mutations in the C-terminal end of the precedinga′ domain led to the identification of several residues that are apparently critical for the assembly of P4H.The interaction between PDI and the α-subunits of the P4H complex and MTP may be related to the interaction between PDI and the incompletely folded substrates on which PDI acts as an isomerase to facilitate folding and native disulfide bond formation. We have demonstrated previously that such misfolded protein substrates bind to PDI, and this binding is competitive with the binding of peptides; by using individual domains and truncated forms of PDI, we showed that theb′ domain constitutes the principal binding site for peptides but that additional domains are implicated in binding misfolded proteins (6.Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (292) Google Scholar).By using chemical cross-linking of a model peptide, we show here that mutations in the C-terminal part of the a′ domain, which affect the assembly of P4H, can also affect the peptide binding activity of PDI, a function associated with the b′ domain. We also demonstrate that the mutations influence the conformation and/or dynamics of the protein and infer that these changes in thea′ domain indirectly influence the binding properties of theb′ domain.DISCUSSIONA key issue in the molecular analysis of PDI is to understand its multifunctionality. In catalyzing protein folding associated with native disulfide bond formation, PDI acts on structured folding intermediates to facilitate disulfide isomerization linked to conformational change (21.Weissman J.S. Kim P.S. Nature. 1993; 365: 185-188Crossref PubMed Scopus (187) Google Scholar, 22.Creighton T.E. Zapun A. Darby N.J. Trends Biotechnol. 1995; 13: 18-23Abstract Full Text PDF PubMed Scopus (89) Google Scholar). As a component of the P4H and MTP complexes its essential function appears to be to interact with nascent or newly synthesized α-subunits to prevent aggregation and maintain an active conformation. What these functions have in common is that PDI appears to interact with incompletely folded polypeptides, although the nature of these interactions is poorly understood.We have monitored the interaction of PDI with such polypeptides directly by cross-linking and have shown that the binding of these substrates is competitive with the binding of shorter unstructured peptides such as the 14-amino acid peptide Δ-somatostatin (20.Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 248: 37-42Crossref PubMed Scopus (84) Google Scholar). From studies with individual PDI domains and linked domain constructs, we demonstrated that the b′ domain forms the essential core of the binding site and is sufficient for the efficient binding of small peptides; fragments without the b′ domain did not show peptide binding (6.Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (292) Google Scholar). The binding properties of the b′ domain for small peptides were identical to the properties observed for purified bovine PDI (20.Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 248: 37-42Crossref PubMed Scopus (84) Google Scholar), i.e. the binding was reversible and sensitive to detergents. The addition of further domains is essential for the binding of more complex substrates, presumably by providing additional binding interactions.The working hypothesis derived from this work is that the peptide-binding site defines the core of a more extended site at which PDI binds its natural substrates for protein folding and possibly also the α-subunits of the P4H and MTP complexes.Although the principal peptide binding site of PDI lies within theb′ domain, it was noted recently that mutations in the C-terminal part of the a′ domain of human PDI affected the assembly into a functional P4H α2β2-tetramer. This clearly raises the question of whether the site of interaction between PDI and the α-subunit of the P4H tetramer is distinct from the peptide-binding site.To address this we investigated by chemical cross-linking the effects of various mutants in the C-terminal part of the a′ domain of recombinant human PDI with respect to peptide binding. This technique can be employed to study specific interactions between proteins available in small amounts without time-consuming purification steps. Model peptide substrates can be cross-linked to PDI and related proteins in the presence of crude cell extracts (23.Klappa P. Stromer T. Zimmermann R. Ruddock L.W. Freedman R.B. Eur. J. Biochem. 1998; 254: 63-69Crossref PubMed Scopus (41) Google Scholar) as well as to purified bovine liver PDI. Cross-linking of model peptides and non-native proteins is specific, saturable, reversible, and independent of the presence of cysteine residues in the bound peptide (20.Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 248: 37-42Crossref PubMed Scopus (84) Google Scholar).Mutations in the C-terminal Part of the a′ Domain of PDI Affect Peptide BindingWe found that the 14-amino acid peptide Δ-somatostatin, after radiolabeling and precipitation, can be chemically cross-linked to wild type PDI and overexpressed in E. coli or Sf9 insect cells. Interestingly, we did not observe any interaction of Δ-somatostatin with endogenous insect PDI. When we compared our results obtained with Sf9 insect cells and E. coli lysates, we found them to be identical, i.e.mutants that showed peptide binding when expressed in Sf9 cells also exhibited peptide binding activity after expression in E. coli cells. Likewise, mutants that failed to bind the peptide in one cell lysate did not show peptide binding in the other cell lysate. We therefore conclude that both cell lysates are compatible with the peptide binding assay, leading to comparable and reproducible results.We observed that mutations in the C-terminal extension of PDI (465–491) or the deletion of this region did not affect the peptide binding activity. However, certain mutations in the precedinga′ domain, specifically all the deletion mutants from residues 436 to 454 and the point mutations F449R and L453E, were completely inactive with respect to peptide binding. There is no current high resolution structure for this domain, but modelling by homology to the a domain suggests that this region comprises a loop (434–442) and the C-terminal α helix (445–454) of thea′ domain. This result is consistent with recent observations that deletion of the entire C-terminal extension has no inhibitory effect on P4H assembly or function or on the disulfide isomerase activity of PDI (16.Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helaakoski T. Notbohm H. Kivirikko K.I. EMBO J. 1999; 18: 65-74Crossref PubMed Scopus (53) Google Scholar), whereas the C-terminal α helix of thea′ domain seems to be a critical region for these functions (Table I). This was also confirmed by recent work of Dai and Wang who showed that truncation of the last 50 amino acids of PDI, comprising part of the C-terminal α helix of the a′ domain, led to inhibition of the chaperone and peptide binding activities (24.Song J. Quan H. Wang C. Biochem. J. 1997; 328: 841-846Crossref PubMed Scopus (28) Google Scholar).Our results clearly demonstrate that mutations in the C-terminal part of the a′ domain that interfere with the assembly of PDI into a functional α2β2 P4H tetramer also affect peptide binding. From this strong correlation we infer that the generation of a functional P4H tetramer requires the peptide-binding site of PDI.Peptide Binding Can Be Restored upon Removal of the Mutated a′ DomainAlthough the CD spectra of the point mutant F449R showed no major differences compared with wild type PDI (16.Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helaakoski T. Notbohm H. Kivirikko K.I. EMBO J. 1999; 18: 65-74Crossref PubMed Scopus (53) Google Scholar), we noticed that this mutant was much more protease-sensitive than the wild type both in cell lysates and when probed by added protease. This clearly indicates that the replacement of phenylalanine 449 by an arginine leads to mobility and/or conformational changes that are not detected by CD spectra and that therefore may be fairly local. Protease treatment of the F449R mutant led to a fragment that co-migrated with thea-b-b′ domain construct of PDI and had the genuine N terminus of PDI. It appears probable that the F449R replacement destabilized the a′ domain, rendering this part of the molecule protease-sensitive. Indeed, structure predictions and homology modeling, based on the structure of the a domain suggest that the side chain of Phe449 is buried in the hydrophobic core of the molecule; replacement by Arg would be expected to have significant effects, whereas substitutions by Tyr and Trp would be more conservative.Once the protease-sensitive a′ domain was removed by treatment with Proteinase K, peptide binding could be restored to the residual a-b-b′ fragment. We therefore infer that the inhibition of peptide binding by mutations in the a′ domain is an indirect effect. Mutations in the a′ domain might induce subtle (but reversible) structural changes in the b′domain, leading to disruption of the peptide-binding site. Alternatively, the altered conformation of the mutated a′domain may physically prevent the reporter peptide from entering the peptide-binding site in the b′ domain. It is also possible that the mutated a′ domain mimics a misfolded polypeptide substrate on which PDI acts. This then would act as a ligand for binding by the peptide binding-site in the b′ domain. Such intramolecular interactions would have a high effective concentration, thus competing strongly for the binding of small peptides. All of the data presented here are fully consistent with the model for intramolecular occupancy of the peptide-binding site, but it should be noted that the F449R mutant shows no major differences in CD spectra to the wild type (16.Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helaakoski T. Notbohm H. Kivirikko K.I. EMBO J. 1999; 18: 65-74Crossref PubMed Scopus (53) Google Scholar).The last two scenarios suggest that the a′ domain is in close proximity to the peptide-binding site in the b′domain, and this model could explain the results obtained previously by Noiva and co-workers (25.Noiva R. Kimura H. Roos J. Lennarz W.J. J. Biol. Chem. 1991; 266: 19645-19649Abstract Full Text PDF PubMed Google Scholar, 26.Noiva R. Freedman R.B. Lennarz W.J. J. Biol. Chem. 1993; 268: 19210-19217Abstract Full Text PDF PubMed Google Scholar), who showed that a radiolabeled tripeptide could be cross-linked to residues within the C-terminal 50 amino acid residues of rat liver PDI (25.Noiva R. Kimura H. Roos J. Lennarz W.J. J. Biol. Chem. 1991; 266: 19645-19649Abstract Full Text PDF PubMed Google Scholar, 26.Noiva R. Freedman R.B. Lennarz W.J. J. Biol. Chem. 1993; 268: 19210-19217Abstract Full Text PDF PubMed Google Scholar) and therefore assigned the peptide-binding site of rat liver PDI to the C terminus of thea′ domain. We speculate that the tripeptide may have bound to the peptide-binding site within the b′ domain and that cross-linking occurred to the C-terminal part of the a′domain, which is in close proximity to the peptide-binding site.From the results presented in this study it is tempting to speculate that there is a common polypeptide-binding site of PDI involved in all its functions. The b′ domain alone being essential and sufficient for the binding of small peptides, but a more extended region involving other domains is required for binding misfolded polypeptides and might be involved in the interaction between PDI and the α-subunit of P4H. Protein-disulfide isomerase (PDI),1 an abundant protein of the eukaryotic endoplasmic reticulum, is a catalyst of disulfide bond formation and rearrangement in the course of protein folding (for review see Ref. 1.Freedman R.B. Klappa P. Bukau B. Molecular Chaperones and Protein Folding. Harwood Academic Press, London1999: 437-459Google Scholar). A molecular interpretation of PDI activity is made difficult by the fact that PDI is multi-functional in the cell. In addition, it has a wide range of actions in vitro and consists of multiple domains. Furthermore, there is as yet no high resolution structure of full-length PDI. Current efforts are focused on analyzing the domain structure of PDI and in establishing the roles of specific domains in specific functional activities. It is now clear (2.Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 3.Freedman R.B. Gane P.J. Hawkins H.C. Hlodan R. McLaughlin S.H. Parry J.W. Biol. Chem. Hoppe-Seyler. 1998; 379: 321-328Crossref PubMed Scopus (51) Google Scholar) that PDI has a structural organization based on duplicated sequence modules (Fig. 1). The full-length protein is constructed of four structural domains with homologous thioredoxin folds plus a C-terminal acidic extension. The homologous a and a′ sequence modules contain the active site motif -WCGHC- and show significant sequence identity to thioredoxin; a high resolution NMR analysis of the recombinanta domain confirms that it has the thioredoxin fold. The homologous b and b′ modules do not show significant sequence similarity to the a domain, but NMR analysis has revealed that the b domain also exhibits the thioredoxin fold (2.Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 4.Kemmink J. Dijkstra K. Mariani M. Scheek R.M. Penka E. Nilges M. Darby N.J. J. Biomol. NMR. 1999; 13: 357-368Crossref PubMed Scopus (68) Google Scholar). Analysis of the properties of individual domains as catalysts of simple thiol:disulfide oxidoreduction and of more complex protein folding linked to disulfide isomerization shows that the a and a′ domains function effectively as simple thiol:disulfide oxidoreductases but that the remaining domains are required for full activity in catalyzing protein folding associated with the formation of native disulfide bonds (5.Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). No specific function has yet been ascribed to the b domain, but cross-linking studies demonstrate that the b′ domain provides the principal peptide-binding site (6.Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (292) Google Scholar). In addition to its role in catalyzing protein folding, PDI has also been described as the β-subunit of two hetero-oligomeric proteins, namely prolyl-4-hydroxylase (P4H) (7.Pihlajaniemi T. Helaakoski T. Tasanen K. Myllyla R. Huhtala M.L. Koivu J. Kivirikko K.I. EMBO J. 1987; 6: 643-649Crossref PubMed Scopus (330) Google Scholar) and the microsomal triglyceride transferase complex (MTP) (8.Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9801-9807Abstract Full Text PDF Google Scholar, 9.Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Biochemistry. 1991; 30: 9728-9735Crossref PubMed Scopus (154) Google Scholar). MTP is obligatory for the assembly of apoB-containing lipoproteins, whereas P4H is important in the post-translational formation of 4-hydroxyproline in collagen in the endoplasmic reticulum (10.Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (235) Google Scholar, 11.Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). It appears, both from attempts at reassociating these complexes after dissociation in vitro(11.Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar) and from studies on assembly of the complexes at biosynthesis, that PDI is required to prevent the aggregation of its partner subunits during either initial folding in the cell or refolding in vitro (12.Vuori K. Pihlajaniemi T. Myllyla R. Kivirikko K.I. EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (127) Google Scholar, 13.Vuori K. Pihlajaniemi T. Marttila M. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7467-7470Crossref PubMed Scopus (114) Google Scholar, 14.John D.C. Grant M.E. Bulleid N.J. EMBO J. 1993; 12: 1587-1595Crossref PubMed Scopus (94) Google Scholar, 15.Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar). Site-directed mutagenesis of the β-subunit/PDI demonstrated that the active site cysteine residues of PDI were not essential for the assembly and activity of active P4H tetramer or of MTP (12.Vuori K. Pihlajaniemi T. Myllyla R. Kivirikko K.I. EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (127) Google Scholar, 15.Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar). The question then arises as to which domains or regions of PDI are involved in or required for its interaction with the other subunits in these hetero-oligomeric complexes. Analysis of deletion and point mutants of PDI recently demonstrated that the acidic C-terminal extension is not critical for the disulfide isomerase activity of PDI or for its ability to assemble into active P4H (16.Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helaakoski T. Notbohm H. Kivirikko K.I. EMBO J. 1999; 18: 65-74Crossref PubMed Scopus (53) Google Scholar). However, deletion and point mutations in the C-terminal end of the precedinga′ domain led to the identification of several residues that are apparently critical for the assembly of P4H. The interaction between PDI and the α-subunits of the P4H complex and MTP may be related to the interaction between PDI and the incompletely folded substrates on which PDI acts as an isomerase to facilitate folding and native disulfide bond formation. We have demonstrated previously that such misfolded protein substrates bind to PDI, and this binding is competitive with the binding of peptides; by using individual domains and truncated forms of PDI, we showed that theb′ domain constitutes the principal binding site for peptides but that additional domains are implicated in binding misfolded proteins (6.Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (292) Google Scholar). By using chemical cross-linking of a model peptide, we show here that mutations in the C-terminal part of the a′ domain, which affect the assembly of P4H, can also affect the peptide binding activity of PDI, a function associated with the b′ domain. We also demonstrate that the mutations influence the conformation and/or dynamics of the protein and infer that these changes in thea′ domain indirectly influence the binding properties of theb′ domain. DISCUSSIONA key issue in the molecular analysis of PDI is to understand its multifunctionality. In catalyzing protein folding associated with native disulfide bond formation, PDI acts on structured folding intermediates to facilitate disulfide isomerization linked to conformational change (21.Weissman J.S. Kim P.S. Nature. 1993; 365: 185-188Crossref PubMed Scopus (187) Google Scholar, 22.Creighton T.E. Zapun A. Darby N.J. Trends Biotechnol. 1995; 13: 18-23Abstract Full Text PDF PubMed Scopus (89) Google Scholar). As a component of the P4H and MTP complexes its essential function appears to be to interact with nascent or newly synthesized α-subunits to prevent aggregation and maintain an active conformation. What these functions have in common is that PDI appears to interact with incompletely folded polypeptides, although the nature of these interactions is poorly understood.We have monitored the interaction of PDI with such polypeptides directly by cross-linking and have shown that the binding of these substrates is competitive with the binding of shorter unstructured peptides such as the 14-amino acid peptide Δ-somatostatin (20.Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 248: 37-42Crossref PubMed Scopus (84) Google Scholar). From studies with individual PDI domains and linked domain constructs, we demonstrated that the b′ domain forms the essential core of the binding site and is sufficient for the efficient binding of small peptides; fragments without the b′ domain did not show peptide binding (6.Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (292) Google Scholar). The binding properties of the b′ domain for small peptides were identical to the properties observed for purified bovine PDI (20.Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 248: 37-42Crossref PubMed Scopus (84) Google Scholar), i.e. the binding was reversible and sensitive to detergents. The addition of further domains is essential for the binding of more complex substrates, presumably by providing additional binding interactions.The working hypothesis derived from this work is that the peptide-binding site defines the core o" @default.
• W2092341922 created "2016-06-24" @default.
• W2092341922 creator A5003895098 @default.
• W2092341922 creator A5016746605 @default.
• W2092341922 creator A5021519057 @default.
• W2092341922 creator A5049252208 @default.
• W2092341922 creator A5053691506 @default.
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• W2092341922 creator A5064900083 @default.
• W2092341922 date "2000-05-01" @default.
• W2092341922 modified "2023-09-29" @default.
• W2092341922 title "Mutations That Destabilize the a′ Domain of Human Protein-disulfide Isomerase Indirectly Affect Peptide Binding" @default.
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