Molecular Characterization of the Principal Substrate Binding Site of the Ubiquitous Folding Catalyst Protein Disulfide Isomerase

Abstract

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b′ domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b′ domain and show the existence of an intradomain linker between the b′ and a′ domains. We have expressed the recombinant b′ domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b′ domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect. Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b′ domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b′ domain and show the existence of an intradomain linker between the b′ and a′ domains. We have expressed the recombinant b′ domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b′ domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect. Native disulfide bond formation in the endoplasmic reticulum is a complex process that is rate-limiting in the biogenesis of many outer membrane and secreted proteins. Native disulfide bond formation can occur via multiple parallel pathways, and there is evidence that a large number of different gene families and redox carriers may play a role in the supply of redox equivalents for protein disulfide bond formation. What is clear is that the rate-limiting step for native disulfide bond formation in proteins that contain multiple disulfides is late-stage isomerization reactions, where disulfide bond formation is linked to conformational changes in protein substrates with substantial regular secondary structure. These steps are thought to be catalyzed only by proteins belonging to the protein disulfide isomerase (PDI) family. PDI 1The abbreviations used are: PDI, protein disulfide isomerase; scRNase, scrambled RNase. was the first catalyst of protein folding identified over 40 years ago (1Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1963; 238: 628-635Abstract Full Text PDF PubMed Google Scholar), but despite probably being the most widely studied protein folding catalyst, significant details of the mechanisms of action of this critical enzyme are still unclear. In all eukaryotes, there exists a species-dependent PDI family of enzymes; for example, in humans (2Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar), ERp72, ERp57, P5, PDIp, PDIr, ERp44 (3Anelli T. Alessio M. Mezghrani A. Simmen T. Talamo F. Bachi A. Sitia R. EMBO J. 2002; 21: 835-844Crossref PubMed Scopus (225) Google Scholar), ERp28/29 (4Ferrari D.M. Nguyen Van P. Kratzin H.D. Söhling H.D. Eur. J. Biochem. 1998; 255: 570-579Crossref PubMed Scopus (75) Google Scholar), ERdj5 (5Cunnea P.M. Miranda-Vizuete A. Bertoli G. Simmen T. Damdimopoulos A.E. Hermann S. Leinonen S. Huikko M.P. Gustafsson J.A. Sitia R. Spyrou G. J. Biol. Chem. 2003; 278: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and ERp18 (6Alanen H.I. Williamson R.A. Howard M.J. Lappi A.K. Jäntti H.P. Rautio S.M. Kellokumpu S. Ruddock L.W. J. Biol. Chem. 2003; 278: 28912-28920Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) have been reported to date. Functional characterization and differentiation between these family members is far from complete. PDI is a multifunctional, multidomain enzyme. The domain structure of PDI has been determined by theoretical (7Ferrari D.M. Söling H-D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (446) Google Scholar) and experimental (8Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (184) Google Scholar, 9Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Freedman R.B. Gane P.J. Hawkins H.C. Hlodan R. McLaughlin S.H. Parry J.W. Biol. Chem. 1998; 379: 321-328Crossref PubMed Scopus (51) Google Scholar, 11Alanen H.I. Salo K.E.H. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W Antiox. Redox Signal. 2003; 5: 367-377Crossref PubMed Scopus (72) Google Scholar) procedures and comprises two catalytic domains, a and a′, separated by two homologous non-catalytic domains, b and b′, plus a C-terminal region designated as c.In addition, it has been proposed (Ref. 11Alanen H.I. Salo K.E.H. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W Antiox. Redox Signal. 2003; 5: 367-377Crossref PubMed Scopus (72) Google Scholar and references therein), but not substantiated, that there is a short linker region between the b′ and a′ domains. In vitro studies indicate that PDI catalyzes all of the steps in native disulfide bond formation but that catalysis of disulfide bond isomerization is most significant over the non-catalyzed rate in a glutathione buffer that mimics the redox potential of the ER. The greatest enhancement of rate is for late-stage isomerizations, i.e. the rate-limiting steps for native disulfide bond formation. PDI is remarkable in that, to our knowledge, it appears to be able to catalyze all of the steps in native disulfide bond formation for all substrate proteins reported. It is still unclear how PDI recognizes all of these different folding states, from essentially unfolded through to substrates with quasinative conformations but lacking specific disulfide bonds, and yet does not appear to interact with correctly folded and disulfide-bonded substrate proteins. Some details have been elucidated of the roles of individual domains of PDI in the different aspects of the overall activity of PDI. It has been reported that oxidation reactions require only a single catalytic domain, simple isomerization reactions require a linear combination of a catalytic domain and the b′ domain, whereas all of PDI excluding the c region is required to catalyze isomerization reactions where disulfide bond formation is linked to conformational changes in protein substrates with substantial regular secondary structure (12Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). The requirement for the non-catalytic b′ domain was subsequently shown to arise due to the fact that the b′ domain contains the principal peptide or non-native protein binding site (13Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar). For small peptide substrates, the b′ domain alone is sufficient for binding, whereas for larger protein substrates, the b′ domain is essential, but other domains contribute to substrate binding. No specificity has yet been reported for substrate binding by PDI, nor has the binding site been located within the b′ domain. However, for the homologue PDIp, the substrate specificity is now well defined (14Ruddock L.W. Freedman R.B. Klappa P. Protein Sci. 2000; 9: 758-764Crossref PubMed Scopus (46) Google Scholar, 15Klappa P. Freedman R.B. Langenbuch M. Lan M.S. Robinson G.K. Ruddock L.W. Biochem. J. 2001; 354: 553-559Crossref PubMed Scopus (34) Google Scholar), a fact that will greatly assist in the localization and definition of the binding site of the PDI family. Here we confirm the existence of the putative linker region between the b′ and a′ domains and report the identification and characterization of the binding site within the b′ domain. Mutations within this site greatly reduce the binding affinity of PDI for small peptide substrates. Secondary Structure Prediction—The sequence of mature human PDI was submitted to the 1996; PubMed Google Scholar). the R. 9: PubMed Scopus Google Scholar) to a for homologous and to a multiple The multiple and this was used as an to The sequence from the multiple was used as an for the secondary structure P. 1997; PubMed Scopus Google Scholar). The secondary structure for the b and b′ domains of human PDI with a different that was the of the b and b′ domains as for the of a multiple sequence and of the PDI b′ of and the a domain of human PDI (8Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (184) Google Scholar), of human and the a′ domain of PDI J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), and of the a and a′ domains of PDI and of the Disulfide of Scholar) to a multiple sequence of E. and human and the and a′ domains of human PDI. In of from different was to the from Creighton and (8Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (184) Google Scholar, 9Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), are on the structural for PDI. The b′ domain of human PDI was to this multiple sequence an of the b and b′ domains a F. PubMed Scopus Google Scholar). The multiple sequence was and secondary structure modeling of the b′ domain was of the homology modeling A. J. Mol. Biol. PubMed Scopus Google Scholar). the with the of the was as the of gene for an for the isolated b′ domain of PDI was by from an PDI H.I. Salo K.E.H. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W Antiox. Redox Signal. 2003; 5: 367-377Crossref PubMed Scopus (72) Google Scholar), that an site to the first of the gene and a site a the The was a of that for an with the The gene the sequence to the first of the domain was as by the the to that there no in the for of on binding and H.I. Salo K.E.H. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W Antiox. Redox Signal. 2003; 5: 367-377Crossref PubMed Scopus (72) Google Scholar), and the for this an The of the is that only can be binding as small effect on significant effect on significant effect on on mature mature mature mature mature mature mature mature mature mature mature mature mature mature mature mature mature mature mature mature mature I272W, mature mature mature b′ domain b′ domain b′ domain b′ domain b′ domain b′ domain b′ domain b′ domain b′ domain b′ domain I272W, domain in a Protein and was in E. in or in and an of for with and protein as the of ERp18 (6Alanen H.I. Williamson R.A. Howard M.J. Lappi A.K. Jäntti H.P. Rautio S.M. Kellokumpu S. Ruddock L.W. J. Biol. Chem. 2003; 278: 28912-28920Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The of protein was determined a PDI b′ domain PDI domain PDI and PDI from E. by of or was as by the was the as (13Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar). on a as an of a with a of a of a of and a of The was for the with no protein on a a as an of with for of a of for the with no protein For the the to the of on protein structure was the of the of to of the for native protein to the over the was it is of and on the of on of the of the b′ of theoretical (7Ferrari D.M. Söling H-D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (446) Google Scholar) and experimental example, 11Alanen H.I. Salo K.E.H. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W Antiox. Redox Signal. 2003; 5: 367-377Crossref PubMed Scopus (72) Google Scholar) studies have defined the domain boundaries for the and a′ domains of the human The domain boundaries of the b′ domain are used in experimental to have defined the boundaries on the of the b domain and the of a b′ domain that is the homologous b domain. in on of PDI R.B. Gane P.J. Hawkins H.C. Hlodan R. McLaughlin S.H. Parry J.W. Biol. Chem. 1998; 379: 321-328Crossref PubMed Scopus (51) Google Scholar), there has been a that a linker region exists between the b′ and a′ domains (2Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar), but there has been no reported experimental of define the domain boundaries of the b′ domain of human PDI, a sequence of the homologous b and b′ domains of human PDI was was the from secondary structure 1996; PubMed Google Scholar) and P. 1997; PubMed Scopus Google Scholar) to and to and of sequence The with and secondary structure is shown in It is clear from this that the b′ domain defined H.I. Salo K.E.H. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W Antiox. Redox Signal. 2003; 5: 367-377Crossref PubMed Scopus (72) Google Scholar) the that an of the linker region between the b′ and a′ domains we as confirm the proposed domain a PDI b′ of mature human was and the of mature human PDI, now with an to be expressed in the of E. and to by a combination of affinity and not The ability to significant of protein in E. from both of these domain that both define the domain boundaries of b′ defined are and region does not of this domain. of the b′ of confirm that the b′ domain is and the activity with it in the full-length a of the b′ domain of PDI was reported to the primary substrate binding site of PDI, binding to small and being for the binding of larger substrates, non-native proteins (13Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar). the b′ and the for ability to bind the peptide via the an E. the expressed the b′ and able to bind as full-length this binding was and on and The far of both that both b′ and well and a significant of and E. and the isolated b domain of PDI the the of these with of the b′ and and to contain that b′ the The b′ a of with the fact that the protein contains and no of a significant in was with no in The contains and of a of of a being in a of the of a of of a for b′ and a single from the native to the in the for and for b′ and The b′ domain of PDI contains two and that may be in the binding affinity on redox potential 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, but R.A. N.J. EMBO J. 2002; 21: PubMed Scopus Google Scholar). on b′ and native and that both as and that both are to in the native was by the of b′ with in the and of not These indicate that of the cysteine are to in substrate binding, nor are to between the and disulfide the of the redox the within the b′ of (13Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar) that the b′ domain of PDI contains the primary substrate binding site of the but there are no on the localization of the substrate binding site within this domain. in this is by the of a structure for PDI or for isolated PDI b′ domain. The of the a and b domains have been by (8Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (184) Google Scholar, 9Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. J. 1999; PubMed Scopus Google Scholar), and both show a b′ is homologous to we to a of b′ on the known structure of b and to use this to the primary substrate binding the structure of the b domain of human PDI has been determined J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. J. 1999; PubMed Scopus Google Scholar), not been the this modeling was only the of E. D.M. J. Mol. Biol. PubMed Scopus Google Scholar), human Biochemistry. PubMed Scopus Google Scholar), and the a domain of human PDI (8Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Biochemistry. 1996; 35: 7684-7691Crossref PubMed Scopus (184) Google Scholar) be used as for homology These with the sequence for the individual domains of PDI, used to a of the b′ domain of PDI the homology modeling A. J. Mol. Biol. PubMed Scopus Google Scholar). the of the domain was the modeled structure for the b′ domain was to that of the b domain with an overall The two cysteine that b′ contains are not in the The two are and are to a this is with the experimental that a of of the cysteine to in the native the substrate specificity of substrate binding by PDI is not there are (13Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar, L.W. Freedman R.B. Klappa P. Protein Sci. 2000; 9: 758-764Crossref PubMed Scopus (46) Google Scholar) that the are in and substrate binding can be by small as the binding specificity of the homologous protein has been reported (14Ruddock L.W. Freedman R.B. Klappa P. Protein Sci. 2000; 9: 758-764Crossref PubMed Scopus (46) Google Scholar, 15Klappa P. Freedman R.B. Langenbuch M. Lan M.S. Robinson G.K. Ruddock L.W. Biochem. J. 2001; 354: 553-559Crossref PubMed Scopus (34) Google Scholar), and it single of and the substrate binding be of the of the b′ domain of PDI no large However, a small be identified to where the site be in the a domain the of and a more be identified the in a of the b′ domain of P. and R. in the non-catalytic domains are thought to have by gene of catalytic domains, it is that the primary substrate binding site in the b′ domain have from the catalytic site and thus be located in the of the These two of evidence the localization of the binding site to the region of the b′ domain of PDI. the within the b′ of the localization of the binding site, a large number of in the putative and in both in the isolated b′ domain and in full-length PDI. These expressed in E. but of the in full-length PDI proteins of the and The was the for no expressed protein be by that this is a significant of the b′ domain was in the and this in to the that no of the expressed in PDI and in the b′ domain for ability to bind The that in the putative peptide binding and whereas in many not was where all and peptide binding, with the greatest effect for single mutation being For a was between the binding properties of in b′ and in full-length PDI, with the of to have a effect in the isolated b′ domain. a was between the ability of proteins to bind and to bind the peptide the on binding more for with the affinity of PDI for this substrate P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; PubMed Scopus Google Scholar). PDI not only small but non-native proteins as we have shown that the b′ domain is for this binding but that it is not with other domains, a and a′, to binding. The ability of the of full-length PDI to bind was by a well The show that this is still able to bind but with affinity with the in the a′ domain of PDI have been shown to peptide binding P. Pirneskoski A. P. J. T. EMBO J. 1999; PubMed Scopus Google Scholar), but this was due to of the a′ domain and the binding of the domain by the b′ domain P. A. Pirneskoski A. P. Ruddock L.W. Freedman R.B. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that the on substrate binding not due to structural and proteins in a of the of full-length PDI expressed in E. for stability that a in stability a structural as for The full-length protein and to by a combination of affinity and not The far of both proteins essentially the of the two proteins different to the of an in the the stability not and for for the These all indicate that the has essentially the structure as the The ability of PDI to as an catalyst of disulfide bond formation in folding polypeptides is on the ability to catalyze disulfide-dithiol exchange, the ability to bind non-native proteins, and the ability to trigger conformational changes in the bound substrate to access to buried cysteine residues. of the has been on exchange, depends on the site through the and many of the reactions that PDI and both site are required for catalysis of native disulfide bond reactions may with the The that between the two play a role in the redox potential of the enzyme J. T. E. P. Freedman R.B. Ruddock L.W. Antiox. Signal. 2003; 5: PubMed Scopus Google Scholar) and thus the of the reactions or have been in the redox potential activity of the J. T. E. P. Freedman R.B. Ruddock L.W. Antiox. Signal. 2003; 5: PubMed Scopus Google Scholar). we have that the changes in the tertiary structure of the isolated catalytic a domain are required to the catalytic A.K. M. H.I. Salo K.E.H. M. A. Ruddock L.W. J. Mol. Biol. Scopus Google Scholar). The other two the ability to bind non-native proteins and the ability to trigger conformational changes in the bound substrate, have It is known that the b′ domain of PDI provides the principal peptide binding site of PDI but that the a and a′ domains contribute to binding of proteins (13Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar). isomerization reactions require a linear combination of catalytic domain plus b′, catalysis of isomerization reactions where disulfide bond is linked to conformational changes in the protein substrate all of PDI excluding the c region (12Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). Here we have defined more precisely the boundaries of the b′ domain, the primary substrate binding site within this domain, and identified to the binding of peptide The of b′ b and the modeling of the b′ structure both that there is a region of between the of the b′ domain and the of the a′ domain, a on the of R.B. Gane P.J. Hawkins H.C. Hlodan R. McLaughlin S.H. Parry J.W. Biol. Chem. 1998; 379: 321-328Crossref PubMed Scopus (51) Google Scholar). We have now expressed this defined b′ domain and shown that it a well defined domain, on the of and the properties and of the recombinant domain. We have not the or region in but the properties of the the and of the that is a region of defined not an the b′ domain as now more the proposed binding site is a small defined by the and of of these peptide binding, with the greatest effect being for substrate binding is to this with the mutation an effect on peptide binding. of full-length PDI and the PDI no of in the structure or stability of the that the is a effect on the substrate binding site and not an structural effect. The effect on the binding of small is the effect on the binding of non-native protein substrates as with the that the a and a′ domains contribute to the binding of proteins (13Klappa P. Ruddock L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (297) Google Scholar). The binding site in the b′ domain is located in a homologous to the of the site in the catalytic domains. is in an within the as in the a domain, whereas is in an to in the a domain. and a that is buried the site in the structure. is to in has been shown to be in catalytic ability M. S. A. Biochemistry. 1997; PubMed Scopus Google Scholar, D.M. C.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is with the that the multidomain structure of PDI through gene of catalytic domains S. T. M. J. Mol. 1998; PubMed Scopus Google Scholar). The small of the putative binding is the specificity for substrate binding for the PDI family has been reported to be a single or with no L.W. Freedman R.B. Klappa P. Protein Sci. 2000; 9: 758-764Crossref PubMed Scopus (46) Google Scholar). studies on the specificity of PDI that a single may the primary but that the is more complex for Pirneskoski and It is that of a non-native protein by this site both from binding a and from with the substrate peptide combination of the two of these be for an unfolded with this the that there is significant potential the binding site in the b′ domain, from both and of the individual roles for potential bond require of binding by multiple substrates in multiple the of the structure of a both of are the of this of the of action of the PDI family is still a but the identification and of the substrate binding site the for of the mechanisms of action of the for the of these to the of as for the of secreted proteins of or from the use of the for We the use of the by the and