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We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of -157 ± 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted folding of plant cyclotides was investigated by comparing the folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct oxidative folding of kalata B1 at physiological pH. A detailed investigation of folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides. We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of -157 ± 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted folding of plant cyclotides was investigated by comparing the folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct oxidative folding of kalata B1 at physiological pH. A detailed investigation of folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides. Protein-disulfide isomerase (PDI 3The abbreviations used are: PDI, protein-disulfide isomerase; ER, endoplasmic reticulum; HPLC, high pressure liquid chromatography; RP-HPLC, reverse phase HPLC; OaPDI, O. affinis PDI; hPDI, human PDI; MS, mass spectrometry; hP5, human P5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 3The abbreviations used are: PDI, protein-disulfide isomerase; ER, endoplasmic reticulum; HPLC, high pressure liquid chromatography; RP-HPLC, reverse phase HPLC; OaPDI, O. affinis PDI; hPDI, human PDI; MS, mass spectrometry; hP5, human P5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.; EC 5.3.4.1) is an oxidoreductase enzyme that belongs to the thioredoxin superfamily (1Ellgaard L. Ruddock L.W. EMBO Rep. 2005; 6: 28-32Crossref PubMed Scopus (626) Google Scholar). It has a major role in oxidative folding of polypeptides in the endoplasmic reticulum (ER) of eukaryotic cells and functions as an ER chaperone (2Wilkinson B. Gilbert H.F. Biochim. Biophys. Acta. 2004; 1699: 35-44Crossref PubMed Scopus (491) Google Scholar). The exact mechanism of action of PDI is not clear, but it is believed to bind polypeptides through hydrophobic interactions and forms (oxidizes), breaks (reduces), and/or shuffles (isomerizes) disulfide bonds in substrate molecules via a dithiol-disulfide exchange between its active-site CXXC motif and the substrate polypeptide (3Gruber C.W. Cemazar M. Heras B. Martin J.L. Craik D.J. Trends Biochem. Sci. 2006; 31: 455-464Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Cyclotides are small disulfide-rich peptides found in plants of the coffee (Rubiaceae) and violet (Violaceae) families (4Craik D.J. Cemazar M. Wang C.K. Daly N.L. Biopolymers. 2006; 84: 250-266Crossref PubMed Scopus (126) Google Scholar). They are typically about 30 amino acids in length and have the unique structural features of a cyclic backbone and a knotted arrangement of three-disulfide bonds, referred to as the cyclic cystine knot motif (5Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (640) Google Scholar). Their compact cyclic cystine knot motif makes them exceptionally resistant to thermal, chemical, or enzymatic degradation (6Colgrave M.L. Craik D.J. Biochemistry. 2004; 43: 5965-5975Crossref PubMed Scopus (448) Google Scholar). Cyclotides exhibit a range of biological activities, including anti-bacterial, cytotoxic, and anti-human immunodeficiency virus activities (7Craik D.J. Science. 2006; 311: 1563-1564Crossref PubMed Scopus (236) Google Scholar), but their natural function is as plant defense molecules (8Gruber C.W. Cemazar M. Anderson M.A. Craik D.J. Toxicon. 2007; 49: 561-575Crossref PubMed Scopus (119) Google Scholar, 9Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (400) Google Scholar). Kalata B1, from the Rubiaceae species Oldenlandia affinis, was the first cyclotide discovered (10Gran L. Medd. Nor. Farm. Selsk. 1970; 12: 173-180Google Scholar), although its macrocyclic structure was not delineated until 1995 (11Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (370) Google Scholar). So far, the sequences of nearly 100 cyclotides have been reported, and it has been suggested that they may surpass the well known plant defensins in number and diversity (12Simonsen S.M. Sando L. Ireland D.C. Colgrave M.L. Bharathi R. Goransson U. Craik D.J. Plant Cell. 2005; 17: 3176-3189Crossref PubMed Scopus (140) Google Scholar, 13Trabi M. Svangard E. Herrmann A. Goransson U. Claeson P. Craik D.J. Bohlin L. J. Nat. Prod. 2004; 67: 806-810Crossref PubMed Scopus (76) Google Scholar). Their unique structural framework, range of bioactivities, and sequence diversity make them interesting targets for pharmaceutical applications (14Craik D.J. Cemazar M. Daly N.L. Curr. Opin. Drug Discov. Devel. 2006; 9: 251-260PubMed Google Scholar). Cyclotides have a characteristic surface-exposed patch of hydrophobic residues that accounts for their late elution on reverse-phase HPLC and membrane binding properties (15Kamimori H. Hall K. Craik D.J. Aguilar M.I. Anal. Biochem. 2005; 337: 149-153Crossref PubMed Scopus (111) Google Scholar). In general, the exposure of a hydrophobic patch on a protein surface is energetically unfavorable, thus requiring special conditions to its In the of cyclotides, the hydrophobic patch is in by the disulfide bonds of the cyclic cystine knot motif it is but the for of the hydrophobic patch is not In a hydrophobic as in the folding of cyclotides N.L. Craik D.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, U. Craik D.J. J. Biol. Full Text Full Text PDF PubMed Scopus (119) Google Scholar), but the mechanism of folding in has until the of cyclotide In this we isolated a novel a protein-disulfide isomerase from the plant O. a and of the cyclotide precursor and the novel PDI (OaPDI), we enzyme activities of the protein with that of human PDI proteins and the to unique sequence features of the plant PDI. We the role of OaPDI in oxidative folding of the cyclotide kalata B1 and a is the first that the oxidative folding of a cyclic cystine knot protein has been PDI. The have in the production of and cyclotides in and in their potential as for pharmaceutical the function and mechanism of the folding PDI. of of OaPDI was isolated reverse OaPDI by of and a on the motif that is in PDI The the and The predicted amino sequences for to PDI sequences and for the presence of the eukaryotic ER the of the sequence was by an O. affinis C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (400) Google and the and The sequence of OaPDI was from the and was to human PDI to the protein sequence and domain and of OaPDI, hPDI, and OaPDI was as protein in a in E. The was the and with and for the The OaPDI protein the sequence of the the the sequence of OaPDI was with the E. cells A was in at until it an of and was with for of at The at 4 to protein by at for The cells in 30 of and and of the the protein was by at for 30 The protein was to protein was to bind to the for with The was a and was protein was of the with and and OaPDI was protein was by on or with as The was by and OaPDI and and mass of the proteins was and protein by and to of and as T. M. 1995; PubMed Scopus Google Scholar, T. M. D. H. T. T. M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. T. J. Biochem. PubMed Scopus Google Scholar). at 4 and used of and mass and/or was on an liquid to a mass with an to a of with 30 at a mass range of with a of and a of 30 was from O. affinis and and reverse for to for and OaPDI and and the O. affinis and by on an and and OaPDI protein and was in the as OaPDI, and the protein was from to the and with with protein was used to in The in the and on protein and used on as by and E. D. a U. S. Scholar). from O. affinis, and in a from human cells used as of and of OaPDI and of the ER sequence was by with the and and the and The an was E. cells The cells and 30 and as for OaPDI to was to and as for OaPDI. was by and was with the as T. K. T. T. R. K. M. Biochem. Biophys. 2005; PubMed Scopus Google Scholar). protein was in at and was and isomerase activity of OaPDI was to the of and Biochem. J. PubMed Scopus Google Scholar). In this the of disulfide bonds in by is with the of to that is by and The of to was by of at as by the of of disulfide bonds, was to Martin J. T. R. A. PubMed Scopus Google Scholar). The of was investigated by the in at and used as and chaperone activities as by the at of and to the for to of and presence of in OaPDI was the of Biochem. Biophys. PubMed Scopus Google Scholar). on a of OaPDI at in conditions in the presence of at to and at to protein structure predicted on the and of of OaPDI at on a in OaPDI was by and by for 30 The redox potential of OaPDI was a redox of protein and of to from to by at and from to in and to for at at the was on a for 30 at The was A. D. S. Biochemistry. 1995; 34: PubMed Scopus Google Scholar), and the redox potential of OaPDI was the at conditions and of kalata B1 was isolated from O. affinis as (5Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (640) Google and by kalata B1 was by in with at for with kalata B1 was on by an in for as N.L. S. Craik D.J. Biochemistry. 1999; PubMed Scopus Google Scholar). of peptides was by and by B1 of and Kalata folding was at in folding from the at between and with and by folding was PDI to and used as of oxidative folding was with of peptides and OaPDI of of of folding intermediates was by mass on a in and by with at for The was with of and Kalata folding in was to for a the was to RP-HPLC, and the was and of on a and as Daly N.L. Waine C. Craik D.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of Kalata B1 and cyclic kalata B1 was at in folding OaPDI for was by and by and activity was an of a C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (400) Google Scholar). on an for and to with kalata B1 OaPDI kalata B1 and a the and the of and was Cyclotides are unique proteins with a arrangement of disulfide bonds, that proteins may be important in their correct folding in the plant are and has been with to their role in folding plant defense we isolated sequences PDI from the Rubiaceae plant O. affinis a with on in known PDI proteins from and surface and of the novel OaPDI and the cyclotide precursor Oak1. folding of the cyclotide kalata B1 and a of this macrocyclic was in RP-HPLC, MS, and We the folding of cyclotide in the presence of PDI to the mechanism of PDI and the of plant cyclotides. We this novel plant PDI by comparing its chaperone and isomerase activities to human PDI proteins and for the first the redox potential of a plant PDI and of the Plant from a of an O. affinis and the OaPDI sequence was used for The for plant PDI and including and The OaPDI the ER amino acids and to a structure features of a PDI. The domain and are with of as in the new PDI is from known in it a of a and the sequence of its domain c has a high predicted in to an acidic domain c in known of OaPDI and of OaPDI and of OaPDI and the precursor protein reverse in a of plant and their OaPDI and are at a similar to a OaPDI was by in O. affinis PDI proteins are in and plants but proteins in a human the protein presence in plant and of OaPDI and at the we their by surface a The the of this was to be and of in OaPDI protein was in E. cells as a protein and a and A and The was by and has a mass of ± The of the PDI was and the number of was of PDI on OaPDI are in the does not the isolated protein was with its in the disulfide The of the and OaPDI by circular of and OaPDI a structure and the was for and a well dramatically the circular and suggested a structure of the protein and the novel PDI and that it was a we it and and of activity of OaPDI was a well on the correct of disulfide bonds in Biochem. J. PubMed Scopus Google Scholar). PDI, human and of proteins have been T. M. D. H. T. T. M. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. T. J. Biochem. PubMed Scopus Google Scholar, T. A. D. T. M. Biochem. J. 2004; PubMed Scopus Google Scholar). PDI and to and a for with the new plant PDI. we the isomerase and chaperone activity of OaPDI with hP5, a of the PDI has isomerase activity than and was used as is from OaPDI and have about and of the isomerase activity of hPDI, activity was in an of of OaPDI chaperone activity to and activity as from The potential of OaPDI was the redox between OaPDI and A and OaPDI was to redox with a redox of known and The of this redox an of and the redox potential of OaPDI was the to be -157 ± 4 is similar to the known redox potential of PDI from that the new plant PDI is functional in chaperone and isomerase and in its redox potential with known PDI it was of to it has a role in the folding of the major protein of O. affinis, cyclotides. Cyclotides are from precursor but it has not been they are to or to cyclotides be in by of N.L. Craik D.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), although it that of a precursor to in we the of PDI on and cyclic forms of the cyclotide kalata of of Cyclotides and cyclic kalata B1 peptides and their and by and the folding was PDI to the of enzyme and for folding a at physiological was for a detailed of folding of and cyclic kalata B1 A and and it was that the PDI has a on in the folding was greater for kalata B1 than for cyclic kalata B1, but the to the correct with a greater The between the in the presence and absence of PDI in the folding was OaPDI, cyclic kalata B1 the disulfide In the presence of OaPDI, the of and cyclic kalata B1 peptides and a and to the potential of we folding in the presence of and as of proteins with PDI In the presence of proteins and in the absence of PDI, the of peptides to and the role of PDI as a was of cyclic and cyclotide derivatives in the presence of OaPDI the of The the of and the the at the of the is as the conditions as or cyclic kalata B1 in a new the and disulfide of the cyclotides, a folding in a hydrophobic that has been to folding of cyclotides in N.L. 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Full Text Full Text PDF PubMed Scopus (119) Google Scholar). the function of the new PDI, folding intermediates in physiological in the absence of OaPDI in the was and at the folding three-disulfide and intermediates in cyclotide folding and of species of from the folding mass is as mass was not and from the folding for peptides are as intermediates intermediates and or cyclic cyclic intermediates cyclic intermediates and cyclic or conditions as kalata B1 OaPDI kalata B1 OaPDI in a new intermediates of folding OaPDI the of species of mass is as to species found to in the for their and number of disulfide bonds is conditions as for kalata B1 kalata B1 in a new In the we have isolated a sequence a new PDI and have that the protein is in a plant that circular defense OaPDI and cyclotide precursor are and the proteins a an in We the new PDI and that it is important for the oxidative folding of and cyclic forms of the cyclotide kalata B1 in OaPDI the of species in and is to disulfide bonds their correct isomerization step is to functional insecticidal cyclotides. The a the mechanism of PDI but the first for oxidative folding of cyclic cystine knot proteins PDI as a from a motif in the a role in oxidoreductase A of OaPDI is that it a in to hPDI, a at the in at this known and the is important for isomerase activity of T. A. D. T. M. Biochem. J. 2004; PubMed Scopus Google Scholar). The protein hP5, a of the in domain has isomerase with in the of the isomerase and a to exchange in in isomerase activity T. A. D. T. M. Biochem. J. 2004; PubMed Scopus Google Scholar). OaPDI exhibits isomerase activity than hPDI, is to the to exchange in the of the for isomerase the in activity in OaPDI is not as as with human proteins T. A. D. T. M. Biochem. 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The of cyclic in the presence of OaPDI was about greater than that of the although the folding was about greater for kalata are with surface in we found that the peptides with OaPDI with greater than cyclic kalata B1 The folding of the and cyclic peptides is by a of and isomerization the cyclic has a than the N.L. S. Craik D.J. Biochemistry. 1999; PubMed Scopus Google Scholar), and thus its folding is energetically the cyclic has a compact structure than the and the of the may be for by PDI, its OaPDI binding and folding it appears that for the the of the cyclic is by the and the of to with PDI. the are the of the is and is essential for the of cyclotides, the of the cyclic accounts for the of cyclic about the role of OaPDI in the oxidative folding of cyclotides, we the major species on folding in the presence and absence of PDI. the of and HPLC elution of the major species and species found for and cyclic kalata B1 and the folding of the and cyclic peptides in the of a folding J.L. S. Trends Biochem. Sci. 2006; 31: Full Text Full Text PDF PubMed Scopus Google Scholar, C. 2006; PubMed Scopus Google Scholar). the peptides are and have their disulfide the in the folding or are in as three-disulfide PDI, the folding to the of disulfide by to the and and peptides are but it appears that they are and are not to disulfide to the in a of In the but the of or intermediates their disulfide is the that isomerization is a major function of this new plant PDI. In a as a hydrophobic that the folding of kalata B1 N.L. Craik D.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, U. Craik D.J. J. Biol. Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The in intermediates conditions of oxidative folding be on the that cyclotides a similar hydrophobic in the presence of an enzyme as they with The structure of PDI S. R. H. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google a hydrophobic in domain cyclotides are to be to this hydrophobic an with the of the correct disulfide bonds be by interactions on the amino sequence of the In the presence of PDI, the disulfide bonds are by dithiol-disulfide exchange between the CXXC motif and the The for the production of cyclotides in is that the disulfide bonds in the precursor are the ER and that and in the A is to be in plant a at the of the cyclotide sequence and a motif that of the in the precursor protein to and the C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (400) Google Scholar). that oxidative folding to in the the potential by the cyclic backbone may not be for in oxidative the for an as PDI, to a surface to the folding In the precursor protein with its J.L. Waine C. Daly N.L. Anderson M.A. Craik D.J. J. 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We have that OaPDI and the precursor on a and a an in of OaPDI the of species in physiological The plant PDI has isomerase but greater chaperone activity than hPDI, is to sequence the motif and in the In cyclotides not their enzyme and the we that a major function of OaPDI is to disulfide bonds their correct to functional insecticidal cyclotides as by The folding of kalata B1 peptides with OaPDI is similar to that in the presence of the hydrophobic to the that hydrophobic on PDI a binding mechanism to in the of the disulfide bonds in the cyclotide kalata The are important for for the of proteins in and in is the of the correct disulfide and is a for to disulfide-rich OaPDI an important for the production of cyclotides in and in Cyclotides have a range of biological activities that make them for and applications as well as novel (14Craik D.J. Cemazar M. Daly N.L. Curr. Opin. Drug Discov. Devel. 2006; 9: 251-260PubMed Google Scholar). the and of the folding OaPDI be used as an in the step for cyclotides or in plants by OaPDI We and for in protein and We for plant and for human protein and for with the We and Martin for
Gruber et al. (Thu,) studied this question.