Circular Proteins in Plants

Much interest has been generated by recent reports on the discovery of circular (i.e. head-to-tail cyclized) proteins in plants. Here we report the three-dimensional structure of one of the newest such circular proteins, MCoTI-II, a novel trypsin inhibitor fromMomordica cochinchinensis, a member of the Cucurbitaceae plant family. The structure consists of a small β-sheet, several turns, and a cystine knot arrangement of the three disulfide bonds. Interestingly, the molecular topology is similar to that of the plant cyclotides (Craik, D. J., Daly, N. L., Bond, T., and Waine, C. (1999) J. Mol. Biol. 294, 1327–1336), which derive from the Rubiaceae and Violaceae plant families, have antimicrobial activities, and exemplify the cyclic cystine knot structural motif as part of their circular backbone. The sequence, biological activity, and plant family of MCoTI-II are all different from known cyclotides. However, given the structural similarity, cyclic backbone, and plant origin of MCoTI-II, we propose that MCoTI-II can be classified as a new member of the cyclotide class of proteins. The expansion of the cyclotides to include trypsin inhibitory activity and a new plant family highlights the importance and functional variability of circular proteins and the fact that they are more common than has previously been believed. Insights into the possible roles of backbone cyclization have been gained by a comparison of the structure of MCoTI-II with the homologous acyclic trypsin inhibitors CMTI-I and EETI-II from the Cucurbitaceae plant family.1IB9 Much interest has been generated by recent reports on the discovery of circular (i.e. head-to-tail cyclized) proteins in plants. Here we report the three-dimensional structure of one of the newest such circular proteins, MCoTI-II, a novel trypsin inhibitor fromMomordica cochinchinensis, a member of the Cucurbitaceae plant family. The structure consists of a small β-sheet, several turns, and a cystine knot arrangement of the three disulfide bonds. Interestingly, the molecular topology is similar to that of the plant cyclotides (Craik, D. J., Daly, N. L., Bond, T., and Waine, C. (1999) J. Mol. Biol. 294, 1327–1336), which derive from the Rubiaceae and Violaceae plant families, have antimicrobial activities, and exemplify the cyclic cystine knot structural motif as part of their circular backbone. The sequence, biological activity, and plant family of MCoTI-II are all different from known cyclotides. However, given the structural similarity, cyclic backbone, and plant origin of MCoTI-II, we propose that MCoTI-II can be classified as a new member of the cyclotide class of proteins. The expansion of the cyclotides to include trypsin inhibitory activity and a new plant family highlights the importance and functional variability of circular proteins and the fact that they are more common than has previously been believed. Insights into the possible roles of backbone cyclization have been gained by a comparison of the structure of MCoTI-II with the homologous acyclic trypsin inhibitors CMTI-I and EETI-II from the Cucurbitaceae plant family.1IB9 reversed-phase high performance liquid chromatography Momordica cochinchinensis two-dimensional total correlation spectroscopy nuclear Overhauser effect two-dimensional NOE spectroscopy double quantum filtered correlation spectroscopy root mean square deviation Over the last few years a number of small disulfide-rich proteins that have a head-to-tail cyclized peptide backbone have been discovered in plants. These circular proteins include kalata B1 (1Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (373) Google Scholar), the circulins (2Gustafson K.R. Sowder R.C., II Henderson L.E. Parsons I.C. Kashman Y. Cardellina II J.H. McMahon J.B. Buckheit Jr., R.W. Pannell L.K. Boyd M.R. J. Am. Chem. Soc. 1994; 116: 9337-9338Crossref Scopus (259) Google Scholar, 3Gustafson K.R. Walton L.K. Sowder R.C.I. Johnson D.G. Pannell L.K. Cardellina J.H.I. Boyd M.R. J. Nat. Prod. 2000; 63: 176-178Crossref PubMed Scopus (100) Google Scholar), and cyclopsychotride A (4Witherup K.M. Bogusky M.J. Anderson P.S. Ramjit H. Ransom R.W. Wood T. Sardana M. J. Nat. Prod. 1994; 57: 1619-1625Crossref PubMed Scopus (228) Google Scholar) from the Rubiaceae family, and various peptides from plants in the Violaceae family (3Gustafson K.R. Walton L.K. Sowder R.C.I. Johnson D.G. Pannell L.K. Cardellina J.H.I. Boyd M.R. J. Nat. Prod. 2000; 63: 176-178Crossref PubMed Scopus (100) Google Scholar, 5Schöpke T. Hasan Agha M.I. Kraft R. Otto A. Hiller K. Sci. Pharm. 1993; 61: 145-153Google Scholar, 6Hallock Y.F. Sowder R.C.I. Pannell L.K. Hughes C.B. Johnson D.G. Gulakowski R. Cardellina J.H.I. Boyd M.R. J. Org. Chem. 2000; 65: 124-128Crossref PubMed Scopus (106) Google Scholar, 7Göransson U. Luijendijk T. Johansson S. Bohlin L. Claeson P. J. Nat. Prod. 1999; 62: 283-286Crossref PubMed Scopus (155) Google Scholar, 8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). They contain ∼30 amino acids, including six highly conserved cysteine residues that form three structurally important disulfide bonds. The latter contribute to the molecules having well defined and stable three-dimensional structures (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). Because of their well defined structures and potent biological activities, the molecules may be regarded as miniproteins. We recently proposed that the known circular proteins most likely form part of a large family of proteins that we refer to as the plant cyclotides (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). They contain the conserved cysteine spacing CX3-CX4-CX4–7-CX1-CX4–5-CX5–7within their circular backbone. The plant cyclotides display a diverse range of biological activities ranging from uterotonic action (kalata B1 (1Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (373) Google Scholar)), to anti-HIV activity (circulins (2Gustafson K.R. Sowder R.C., II Henderson L.E. Parsons I.C. Kashman Y. Cardellina II J.H. McMahon J.B. Buckheit Jr., R.W. Pannell L.K. Boyd M.R. J. Am. Chem. Soc. 1994; 116: 9337-9338Crossref Scopus (259) Google Scholar)), to hemolytic activity (5Schöpke T. Hasan Agha M.I. Kraft R. Otto A. Hiller K. Sci. Pharm. 1993; 61: 145-153Google Scholar, 9Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8913-8918Crossref PubMed Scopus (406) Google Scholar, 10Daly N.L. Love S. Alewood P.F. Craik D.J. Biochemistry. 1999; 38: 10606-10614Crossref PubMed Scopus (194) Google Scholar), and inhibition of neurotensin binding (cyclopsychotride A (4Witherup K.M. Bogusky M.J. Anderson P.S. Ramjit H. Ransom R.W. Wood T. Sardana M. J. Nat. Prod. 1994; 57: 1619-1625Crossref PubMed Scopus (228) Google Scholar)). Although their precise role in plants has not yet been reported, it appears that they are most likely present as defense molecules. All of their biological activities in mammalian systems seem to be related in one way or another to membrane interactions, a common feature of plant defense molecules. Recently the cyclotides have also been reported to display a wide range of anti-microbial activities against Gram-positive and Gram-negative bacteria, and against certain fungi (9Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8913-8918Crossref PubMed Scopus (406) Google Scholar), further enhancing the likelihood that they are indeed plant defense molecules. The three-dimensional structures of three members of the plant cyclotide family have now been determined, including the prototypic member kalata B1, the first macrocyclic peptide for which a full three-dimensional structure was determined (1Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (373) Google Scholar), circulin A (11Daly N.L. Koltay A. Gustafson K., R. Boyd M.R. Casas-Finet J.R. Craik D.J. J. Mol. Biol. 1999; 285: 333-345Crossref PubMed Scopus (114) Google Scholar), and cycloviolacin O1 (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). The structures are all highly conserved and contain a distorted triple stranded β-sheet, perhaps better described as a well defined β-hairpin with a third strand forming a β-bulge (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). The three disulfide bonds are arranged in a so called cystine knot, in which an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments forms a ring that is penetrated by the third disulfide bond (12McDonald N.Q. Hendrickson W.A. Cell. 1993; 73: 421-424Abstract Full Text PDF PubMed Scopus (475) Google Scholar, 13Isaacs N.W. Curr. Opin. Struct. Biol. 1995; 5: 391-395Crossref PubMed Scopus (180) Google Scholar, 14Pallaghy P.K. Nielsen K.J. Craik D.J. Norton R.S. Protein Sci. 1994; 3: 1833-1839Crossref PubMed Scopus (469) Google Scholar, 15Craik D.J. Daly N.L. Waine C. Toxicon. 2001; 39: 43-60Crossref PubMed Scopus (417) Google Scholar). Although a seemingly unlikely structural motif, the cystine knot has now been seen in a variety of small disulfide-rich proteins from both the plant and animal kingdoms (15Craik D.J. Daly N.L. Waine C. Toxicon. 2001; 39: 43-60Crossref PubMed Scopus (417) Google Scholar), and seems to be associated with exceptionally high stability and resistance to proteolytic cleavage of molecules containing it. This is particularly so for the cyclotides, which contain a cyclic cystine knot motif (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). Recently Hernandez et al. (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar) reported two new macrocyclic peptides from Momordica cochinchinensis, a plant from the Cucurbitaceae plant family. The two and MCoTI-II are trypsin inhibitors and amino acids, a cyclized peptide backbone, and three disulfide bonds. They are homologous to a large range of trypsin inhibitors from plants (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar), are the first reported macrocyclic trypsin inhibitors that a cyclic trypsin inhibitor was recently reported from S. R.S. J. Mol. Biol. 1999; Scopus Google Scholar)). This discovery that macrocyclic peptides are more common in plants than has previously been and that more are likely to be discovered The of the two new trypsin and MCoTI-II, with the known plant cyclotides from the cysteine residues and the macrocyclic peptide backbone, as seen by a comparison of MCoTI-II and kalata B1 in that the molecules are cyclic the of of is the we have the by al. (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar). we for the six cysteine residues and the six backbone that the the cysteine residues This is defined in The three-dimensional structure of MCoTI-II has been (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar) on to trypsin of the three-dimensional structure of the new macrocyclic peptides is We to class was similar to the plant cyclotides and the cystine knot We and a of MCoTI-II from M. cochinchinensis and report the three-dimensional structure determined spectroscopy and of the for structures of class of molecules is that they are known to be highly stable in the of kalata B1, are a most feature for molecules. This is in the of the plant from which kalata B1 is of certain in the a from the plant to L. Sletten K. J. 2000; PubMed Scopus Google Scholar). The of the cyclic cystine knot macrocyclic disulfide as a in is D. J., Daly, N. L., and K. J. Scholar). The three-dimensional structure reported a for as well as that the new trypsin inhibitors form part of the cyclotide family. cochinchinensis from a in The a and with The was a was to the a of was a which the was to and for The was a on a of and with a of and the was on a The was with and for peptide in or and on a The two-dimensional similar to for the related cyclic peptide circulin A (11Daly N.L. Koltay A. Gustafson K., R. Boyd M.R. Casas-Finet J.R. Craik D.J. J. Mol. Biol. 1999; 285: 333-345Crossref PubMed Scopus (114) Google Scholar) and and a with a of and with of and by a of and of the peptide in from from the to K. to given in Daly et al. (11Daly N.L. Koltay A. Gustafson K., R. Boyd M.R. Casas-Finet J.R. Craik D.J. J. Mol. Biol. 1999; 285: 333-345Crossref PubMed Scopus (114) Google Scholar). to and the and C. P. M. K. J. PubMed Scopus Google Scholar). and from with with to for than and for than and for residues in an in with NOE residues in the on The of the residues on the in the bonds determined on and structure bond of and for the and structures P. C. K. J. Mol. Biol. PubMed Scopus Google Scholar) to NOE for and and bond in structure The structures generated a 1994; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar) the Scholar). of structures was the with of M. J. Mol. Biol. PubMed Scopus Google Scholar) in a on the D.J. S. M. J. Chem. Scopus Google Scholar). Protein Sci. 5: PubMed Scopus Google Scholar) and R. J. PubMed Scopus Google Scholar). The of M. cochinchinensis with as described previously (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar) and the was peptides and their molecular by with the previously macrocyclic and MCoTI-II present in to to the and forms of MCoTI-II (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar). the (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar) the peptide bond and was to be to and and was in the of MCoTI-II and to with was as the systems in and the in with of the (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar). K. of and Google Scholar) and the are as The of a and of MCoTI-II are given in The is well and the The and large number of in the also the first of a well defined three-dimensional the of MCoTI-II has an molecular to the spectroscopy was to molecules. one form on the NOE residues and which form the peptide backbone. of the of both that the molecules have similar with the of the residues and the of the two in the we have determined the three-dimensional structure of the of MCoTI-II with the of the NOE and the structure to be as in A β-hairpin is present residues and are and residues of (i.e. the and A. R.S. J. 1995; 5: PubMed Scopus Google of the structure and the of The three-dimensional structure of MCoTI-II was determined the Scholar). The disulfide not been determined and so it was important to small disulfide-rich molecules in which the disulfide bonds form a such an is the one to the of cleavage structure disulfide bonds as with the disulfide to in trypsin inhibitors D. J. T. J. Mol. Biol. PubMed Scopus Google Scholar) previously and in the cyclotide peptides (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar) and This was on an of all possible of in the structures This that the to the and structure with the disulfide bonds in way and structures of and with the with the homologous inhibitors and the plant cyclotides was as for the proposed disulfide bonds also from the structure in with the and all with the in The family of structures for MCoTI-II is well defined most of the are few or range for the first residues and in a of in This is in the that are for the and for residues and for residues and that the structures are given as The for residues are and for the backbone and all The are and for residues The of the three-dimensional structure are a and several of the structure in the family of structures with Protein Sci. 5: PubMed Scopus Google Scholar) a residues and and residues and A β-hairpin is by residues and as from the structure and a residues and the The the and residues are not by as part of a However, is also for related small disulfide-rich peptides and is not the plant cyclotides D. J., Daly, N. L., and K. J. Scholar) the third is to the well defined the of and of the of the in the structure are such that they are not as part of a in all of the The disulfide bonds of MCoTI-II form a cystine structure similar to that in the plant cyclotides. arrangement the embedded ring formed by and and their connecting backbone segments is by the third disulfide A of the structure and the cystine knot of MCoTI-II is given in the peptide backbone of MCoTI-II is a way to the structural is in of the six or that are by of the backbone in way and highlights several of the including the of the three and the various The disulfide bond is also and II a highly on with acyclic trypsin the residues two and two and a residues and a residues and residues a a amino and is one of the that the is by which a and into which the strand of the the structures the disulfide bonds the circular backbone and and are to one from the structure more than of the and is also the most that structures in their of to the of the This may or a of a high and the number of possible and is to amino it was of or that in the for the of the of various amino was are associated with in can be of in an of acyclic of kalata B1, with on a for cysteine N.L. Craik D.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the for the six cysteine residues of MCoTI-II the of in The disulfide bond connecting residues to is the disulfide bond to the and the are for both of in of the the for the cysteine residues are by to and to well defined of the The in was in the determined by Hernandez et al. (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar). The also and and indeed are residues in However, the possible and was in few of the A comparison of MCoTI-II with the trypsin inhibitor CMTI-I D. J. T. J. Mol. Biol. PubMed Scopus Google Scholar) and the plant kalata B1, is given in is the proteins similar the backbone of the cysteine residues of MCoTI-II and CMTI-I with an of The for kalata B1 and MCoTI-II is of the in the cysteine However, the backbone of the cysteine residues that the ring of the cystine knot the is trypsin EETI-II A. Chiche L. D. Biochemistry. PubMed Scopus Google Scholar), also has a similar structure to MCoTI-II and with an of the backbone of the cysteine the we have determined the three-dimensional structure of MCoTI-II, a trypsin inhibitor from M. has the feature of a head-to-tail cyclized peptide backbone, and the of an number of small circular proteins discovered the last The of structure is a β-hairpin and associated cystine knot A third strand of is with and the also several The is similar to that of the trypsin inhibitors EETI-II and have high with MCoTI-II are proteins in that they the head-to-tail cyclization of The of the of the cyclic inhibitor to of the of is the role of be including to or in of new or from proteolytic We in and discovered cyclic plant with the previously reported cyclotide family (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar) of macrocyclic of cyclization include of the residues into a for or associated with the of The of small peptides are and contribute to on binding to proteins. can such by the However, not seem to be the role of cyclization in the of MCoTI-II, the in cyclization is in fact the for MCoTI-II has yet to be reported, the peptide is likely to be from a of which in the of and to form This is on with the for the related peptide from J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), is with forming a of the peptide an it is that the is highly and in the cyclic The that the in than a of is by an of of that not the role of cyclization it was of interest to be associated with cyclization that are important in a than in a binding has been that the of inhibition of the peptides is to that for trypsin on the in the inhibitory (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar). The of the residues to be the are in for MCoTI-II and a range of both in and to of a CMTI-I D. J. T. J. Mol. Biol. PubMed Scopus Google Scholar), with the cyclic MCoTI-II similar backbone in with the most in the of the and to a in the of the The structures of MCoTI-II and CMTI-I are also similar to the structures in with the of the of the The of a MCoTI-II and in or that cyclization not a role in the of MCoTI-II and trypsin structures determined with inhibitors to The Protein are CMTI-I CMTI-I The structures determined with inhibitors to The Protein are CMTI-I D. J. T. J. Mol. Biol. PubMed Scopus Google S. Y. J. Mol. Biol. 1993; PubMed Scopus Google CMTI-I R. J. T. PubMed Scopus Google R. J. Biol. 1999; PubMed Scopus Google S. R.S. J. Mol. Biol. 1999; Scopus Google S. J.P. 5: Full Text Full Text PDF PubMed Google Scholar). in a new The inhibitor in trypsin inhibitor from the in that it is a disulfide bond and all MCoTI-II, is cyclic S. R.S. J. Mol. Biol. 1999; Scopus Google Scholar). is a particularly potent trypsin inhibitor small and an to the residues for three-dimensional structure in with has been determined S. R.S. J. Mol. Biol. 1999; Scopus Google Scholar) and the residues are in similar to for the inhibitors The fact that potent trypsin inhibition can be with such a small inhibitor is with the fact that of the MCoTI-II inhibitor are not for binding activity and and or roles for the have been and to or of can be possible role for cyclization is that of from proteolytic on the which that or roles are seems the most likely of cyclization in of small peptides is a well in the to small peptides against cleavage a the MCoTI-II peptide most likely has to for to and be by MCoTI-II has also been to be to the (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar). This is with the plant kalata B1, which is to a range of including and (1Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (373) Google Scholar). the cyclization of macrocyclic peptides appears to be an of as is the for small B1 is one member of is now a large family of cyclotides (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). Although MCoTI-II is of similar amino and cystine it to the previously plant cyclotides. the three-dimensional structure is and of structure and the cystine knot The of a cyclic peptide backbone and a cystine knot motif that MCoTI-II into the cyclic cystine knot structural (8Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar, 15Craik D.J. Daly N.L. Waine C. Toxicon. 2001; 39: 43-60Crossref PubMed Scopus (417) Google Scholar). The structural the that the cyclotides and the Momordica peptides may be the fact that they have different biological the of different biological activities for the various members of the cyclotide family of the that the cyclic cystine knot is particularly stable and to of a range of different (15Craik D.J. Daly N.L. Waine C. Toxicon. 2001; 39: 43-60Crossref PubMed Scopus (417) Google Scholar). that the of the cystine knot motif in peptides is different from of the known cyclotides and that the embedded ring in the structure amino than This the cystine knot the family of structures are well defined the of the cystine the associated with backbone cyclization appears more in the macrocyclic trypsin inhibitors than in the previously reported plant cyclotides. the the consists of small amino acids, in the plant cyclotides the residues are The previously reported cyclotides are by their on This is to from a of residues (1Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (373) Google Scholar). the of kalata B1 we have that of the disulfide N.L. Love S. Alewood P.F. Craik D.J. Biochemistry. 1999; 38: 10606-10614Crossref PubMed Scopus (194) Google Scholar). The macrocyclic peptides from than the cyclotides, The peptides also contain more particularly than the cyclotides. The of and residues for MCoTI-II is in of the residues are as a of the of the containing the disulfide bonds and for These residues on one of the The of the most of the and with the residues of and residues has previously been to be for antimicrobial The of residues on the of MCoTI-II that such an activity may be possible for This is by the report that a from M. cochinchinensis which MCoTI-II, antimicrobial (16Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. J.P. A. T. Nguyen D. Biochemistry. 2000; 39: Scopus Google Scholar). the previously reported cyclotides and the circular peptides is the of for the cyclotides. inhibitors have been known for so has been for forms of the cyclotides. This may be a of the that have been to or may highly of the cyclotides from MCoTI-II and the plant cyclotides the similar three-dimensional structure and cyclic backbone they can be regarded as part of the family of proteins. the cyclotide family can be to include the new cyclic trypsin inhibitors and This expansion of the cyclotide family to include trypsin inhibitors from the Cucurbitaceae family highlights the importance and functional variability of The of of circular proteins with the associated with the of for the of circular proteins M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, H. 2000; PubMed Scopus Google Scholar, Jr., J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. A. 1999; PubMed Scopus Google Scholar). We the for a of the with