An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides.
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Four macrocyclic cystine-knot peptides of 29-31 residues, kalata, circulin A and B (CirA and CirB), and cyclopsychotride, have been isolated from coffee plants but have undetermined physiological functions. These macrocycles and 10 of their analogs prepared by chemical synthesis were tested against nine strains of microbes. Kalata and CirA were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of approximately 0.2 microM. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, CirB and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, CirB showed potent activity against E. coli with a minimum inhibitory concentration of 0.41 microM. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans. These macrocycles are cytotoxic and lysed human red blood cell with a lethal dose 50% of 400 microM. Modifying the Arg residue in kalata with a keto aldehyde significantly reduced its activity against S. aureus whereas blocking the arg in CirA produced no significant effect. The two-disulfide variants and their scrambled disulfide isomers exhibited antimicrobial profiles and potency similar to their native peptides. However, in high-salt assays (100 mM NaCl), few of these macrocyclic peptides, natives or analogs, retained antimicrobial activity. These results show that the macrocyclic peptides possess specific and potent antimicrobial activity that is salt-dependent and that their initial interactions with the microbial surfaces may be electrostatic, an effect commonly found in defensin antimicrobial peptides. Furthermore, their end-to-end cyclic structure with a cystine-knot motif represents a molecular structure of antimicrobials and may provide a useful template for the design of novel peptide antibiotics. (+info)
Acyclic permutants of naturally occurring cyclic proteins. Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1.
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Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple-stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the beta-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity. (+info)
Cyclotides: a novel type of cytotoxic agents.
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Cytotoxic activities of three naturally occurring macrocyclic peptides (cyclotides) isolated from the two violets, Viola arvensis Murr. and Viola odorata L., were investigated. A nonclonogenic fluorometric microculture assay was used to examine cytotoxicity in a panel of 10 human tumor cell lines representing defined types of cytotoxic drug resistance. Additionally, primary cultures of tumor cells from patients, and for comparison normal lymphocytes, were used to quantify cytotoxic activity. All three cyclotides, varv A, varv F, and cycloviolacin 02, exhibited strong cytotoxic activities, which varied in a dose-dependent manner. Cycloviolacin 02 was the most potent in all cell lines (IC50 0.1-0.3 microM), followed by varv A (IC50 2.7-6.35 microM) and varv F (IC50 2.6-7.4 microM), respectively. Activity profiles of the cyclotides differed significantly from those of antitumor drugs in clinical use, which may indicate a new mode of action. This, together with the exceptional chemical and biological stability of cyclotides, makes them interesting in particular for their potential as pharmacological tools and possibly as leads to antitumor agents. (+info)
Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides.
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The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (Le-Nguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway. (+info)
The CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto. Functional and structural insights through disulfide structure analysis.
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The disulfide structure of the CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto protein was determined by a combination of enzymatic and chemical fragmentation, followed by chromatographic separation of the fragments, and characterization by mass spectrometry and N-terminal sequencing. These studies showed that Cys115 forms a disulfide bond with Cys133, Cys128 with Cys149, and Cys131 with Cys140. Protein database searching and molecular modeling revealed that the pattern of disulfide linkages in the CFC domain of Cripto is the same as that in PARS intercerebralis major Peptide C (PMP-C), a serine protease inhibitor, and that the EGF-CFC domains of Cripto are predicted to be structurally homologous to the EGF-VWFC domains of the C-terminal extracellular portions of Jagged 1 and Jagged 2. Biochemical studies of the interactions of ALK4 with the CFC domain of Cripto by fluorescence-activated cell sorter analysis indicate that the CFC domain binds to ALK4 independent of the EGF domain. A molecular model of the CFC domain of Cripto was constructed based on the nuclear magnetic resonance structure of PMP-C. This model reveals a hydrophobic patch in the domain opposite to the presumed ALK4 binding site. This hydrophobic patch may be functionally important for the formation of intra or intermolecular complexes. (+info)
Disulfide mapping of the cyclotide kalata B1. Chemical proof of the cystic cystine knot motif.
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The cyclotides are a recently discovered family of plant proteins that have the fascinating structural feature of a continuous cyclic backbone and, putatively, a knotted arrangement of their three conserved disulfide bonds. We here show definite chemical proof of the I-IV, II-V, III-VI knotted disulfide connectivity of the prototypic cyclotide kalata B1. This has been achieved by a new approach for disulfide analysis, involving partial reduction and stepwise alkylation including introduction of charges and enzymatic cleavage sites by aminoethylation of cysteines. The approach overcomes the intrinsic difficulties for disulfide mapping of cyclotides, i.e. the cyclic amide backbone, lack of cleavage sites between cysteines, and a low or clustered content of basic amino acids, and allowed a direct determination of the disulfide bonds in kalata B1 using analysis by mass spectrometry. The established disulfide connectivity is unequivocally shown to be cystine knotted by a topological analysis. This is the first direct chemical determination of disulfides in native cyclotides and unambiguously confirms the unique cyclic cystine knot motif. (+info)
A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation.
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The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mm, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe6, and the second involving a charge-charge interaction with the Asp25 residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules. (+info)
Solution structure of the cyclotide palicourein: implications for the development of a pharmaceutical framework.
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The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities. (+info)