A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides.

A common structural motif consisting of a cystine knot and a small triple-stranded beta-sheet has been defined from comparison of the 3-dimensional structures of the polypeptides omega-conotoxin GVIA (Conus geographus), kalata BI (Oldenlandia affinis DC), and CMTI-I (Curcurbita maxima). These 3 polypeptides have diverse biological activities and negligible amino acid sequence identity, but each contains 3 disulfide bonds that give rise to a cystine knot. This knot consists of a ring formed by the first 2 bonds (1-4 and 2-5) and the intervening polypeptide backbone, through which the third disulfide (3-6) passes. The other component of this motif is a triple-stranded, anti-parallel beta-sheet containing a minimum of 10 residues, XXC2, XC5X, XXC6X (where the numbers on the half-cysteine residues refer to their positions in the disulfide pattern). The presence in these polypeptides of both the cysteine knot and antiparallel beta-sheet suggests that both structural features are required for the stability of the motif. This structural motif is also present in other protease inhibitors and a spider toxin. It appears to be one of the smallest stable globular domains found in proteins and is commonly used in toxins and inhibitors that act by blocking the function of larger protein receptors such as ion channels or proteases.     

Processing of a 22 kDa precursor protein to produce the circular protein tricyclon A

Cyclotides are a family of plant proteins that have the unusual combination of head-to-tail backbone cyclization and a cystine knot motif. They are exceptionally stable and show resistance to most chemical, physical, and enzymatic treatments. The structure of tricyclon A, a previously unreported cyclotide, is described here. In this structure, a loop that is disordered in other cyclotides forms a beta sheet that protrudes from the globular core. This study indicates that the cyclotide fold is amenable to the introduction of a range of structural elements without affecting the cystine knot core of the protein, which is essential for the stability of the cyclotides. Tricyclon A does not possess a hydrophobic patch, typical of other cyclotides, and has minimal hemolytic activity, making it suitable for pharmaceutical applications. The 22 kDa precursor protein of tricyclon A was identified and provides clues to the processing of these fascinating miniproteins.     

Diversity in the disulfide folding pathways of cystine knot peptides

Summary The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.     

A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle

The inhibitor cystine knot (ICK) structural motif has been found in several small proteins and peptides from plants, insects, marine molluscs, and also in human. It is defined by a triple beta-sheet that is held together by three intramolecular disulfide bonds built by six conserved cysteine residues that generate a highly rigid and stable fold. We describe a procedure for the production of ICK peptides with correct disulfide bond connectivities via expression in Escherichia coli as fusion proteins with an enzymatically inactive variant of the Bacillus amyloliquefaciens RNAse barnase. Barnase directs the fused peptide to the culture medium and the fusion protein can be isolated by combined cation exchange/reverse-phase chromatography. The ICK peptides are released from the barnase expression and purification handle either by cyanogen bromide or by protease cleavage to give pure and correctly folded cystine knot peptides.     

The cyclotide family of circular miniproteins: nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The structural features that contribute to their remarkable stability are described in this review.     

Disulfide mapping of the cyclotide kalata B1. Chemical proof of the cystic cystine knot motif

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.     

Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides

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.     

Formation of Cystine Slipknots in Dimeric Proteins

We consider mechanical stability of dimeric and monomeric proteins with the cystine knot motif. A structure based dynamical model is used to demonstrate that all dimeric and some monomeric proteins of this kind should have considerable resistance to stretching that is significantly larger than that of titin. The mechanisms of the large mechanostability are elucidated. In most cases, it originates from the induced formation of one or two cystine slipknots. Since there are four termini in a dimer, there are several ways of selecting two of them to pull by. We show that in the cystine knot systems, there is strong anisotropy in mechanostability and force patterns related to the selection. We show that the thermodynamic stability of the dimers is enhanced compared to the constituting monomers whereas machanostability is either lower or higher.     

Factors Influencing the Stability of Cyclotides: Proteins with a Circular Backbone and Cystine Knot Motif

Cyclotides are a large family of mini-proteins that have the distinguishing features of a head-to-tail cyclised backbone and a cystine knot formed by six conserved cysteine residues. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families. The unique structural features of the cyclotides make them extremely resistant to chemical, thermal and proteolytic degradation. In this article we review recent studies from our laboratory that dissect the role of the individual structural elements in defining the stability of cyclotides. The resistance of cyclotides to chemical and proteolytic degradation is in large part due to the cystine knot, whereas the thermal stability is a composite of several features including the cystine knot, the cyclic backbone and the hydrogen bonding network. A range of biological activities of cyclotides is critically dependent on the presence of the cyclic backbone.Australian Peptide Conference Issue.     

Cystine knot mutations affect the folding of the glycoprotein hormone alpha-subunit. Differential secretion and assembly of partially folded intermediates

The common glycoprotein hormone alpha-subunit (GPH-alpha) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10-60, 28-82, and 32-84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-alpha folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin beta-subunit (hCG-beta). Mutation of non-cystine knot disulfide bond 7-31, bond 59-87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-beta. From these studies we conclude that: (i) the cystine knot of GPH-alpha is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-alpha folding, its ability to be secreted, and its ability to heterodimerize with hCG-beta.     

Oxidative folding of the cystine knot motif in cyclotide proteins

The cyclotides are a large family of plant proteins that have a cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Despite the apparent complexity of their cystine knot motif it is possible to efficiently fold these proteins, as exemplified by oxidative folding studies on the prototypic cyclotide, kalata B1. This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates.     

Intracellular folding pathway of the cystine knot-containing glycoprotein hormone alpha-subunit

Three of the five disulfide bonds in the glycoprotein hormone alpha-subunit (GPH-alpha) form a cystine knot motif that stabilizes a three-loop antiparallel structure. Previously, we described a mutant (alpha(k)) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-alpha folding and secretion. In this study, we used alpha(k) as a model to study the intracellular GPH-alpha folding pathway. Cystine knot formation proceeded through a 1-disulfide intermediate that contained the 28-82 disulfide bond. Formation of disulfide bond 10-60, then disulfide bond 32-84, followed the formation of 28-82. Whether the two non-cystine knot bonds 7-31 and 59-87 could form independent of the knot was also tested. Disulfide bond 7-31 formed rapidly, whereas 59-87 did not form when all cysteine residues of the cystine knot were converted to alanine, suggesting that 7-31 forms early in the folding pathway and that 59-87 forms during or after cystine knot formation. Finally, loop 2 of GPH-alpha has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-alpha folding. To test this, we replaced residues 36-55 in the flexible loop 2 with an artificially flexible glycine chain. Consistent with our hypothesis, folding and secretion were unaffected when loop 2 was replaced with the glycine chain. Based on these findings, we describe a model for the intracellular folding pathway of GPH-alpha and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.     

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX₃CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.     

Circular proteins in plants: solution structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis

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 from Momordica cochinchinensis, a member of the Cucurbitaceae plant family. The structure consists of a small beta-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.     

Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules

The cystine knot three-dimensional structure is found in many extracellular molecules and is conserved among divergent species. The identification of proteins with a cystine knot structure is difficult by commonly used pairwise alignments because the sequence homology among these proteins is low. Taking advantage of complete genome sequences in diverse organisms, we used a complementary approach of pattern searches and pairwise alignments to screen the predicted protein sequences of five model species (human, fly, worm, slime mold, and yeast) and retrieved proteins with low sequence homology but containing a typical cystine knot signature. Sequence comparison between proteins known to have a cystine knot three-dimensional structure (transforming growth factor-beta, glycoprotein hormone, and platelet-derived growth factor subfamily members) identified new crucial amino acid residues (two hydrophilic amino acid residues flanking cysteine 5 of the cystine knot). In addition to the well known members of the cystine knot superfamily, novel subfamilies of proteins (mucins, norrie disease protein, von Willebrand factor, bone morphogenetic protein antagonists, and slit-like proteins) were identified as putative cystine knot-containing proteins. Phylogenetic analysis revealed the ancient evolution of these proteins and the relationship between hormones [e.g. transforming growth factor-beta (TGFbeta)] and extracellular matrix proteins (e.g. mucins). They are absent in the unicellular yeast genome but present in nematode, fly, and higher species, indicating that the cystine knot structure evolved in extracellular signaling molecules of multicellular organisms. All data retrieved by this study can be viewed at http://hormone.stanford.edu/.     

Knots in rings. The circular knotted protein Momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys1-Cys18 disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.     

Identifying DNA-binding proteins using structural motifs and the electrostatic potential

Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif.     

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata B2, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.     

Acyclic permutants of naturally occurring cyclic proteins. Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

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.     

Solution structure of the carboxyl-terminal cysteine-rich domain of the VHv1.1 polydnaviral gene product: comparison with other cystine knot structural folds.

Polydnaviruses are an unusual group of insect viruses that have an obligate symbiotic association with certain parasitic wasps. These viruses are transmitted with the wasp egg during oviposition into lepidopteran insects, enabling the survival and development of the egg inside the host larvae. We report the three-dimensional structure of a novel polydnaviral cysteine-rich motif (cys-motif), identified as the carboxyl-terminal domain of a two cys-motif containing polydnaviral VHv1.1 gene product, abbreviated "C-term VHv1.1". This 65-residue domain was identified experimentally by limited proteolysis of the full-length protein and was subsequently cloned in a bacterial expression system for NMR studies. The C-term VHv1.1 3D structure was determined in solution by two-dimensional (1)H NMR spectroscopy. Calculation of the structure was based on a total of 300 upper distance restraints and 20 dihedral angle constraints, and resulted in an ensemble of 25 representative conformers with an average rmsd of 0.47 A from the mean structure for core backbone atoms. The protein core is made of a four beta-strand scaffold held together in a compact structure by three disulfide bonds, which form a cystine knot. The four beta-strands are arranged in an unusual configuration to form a triple-stranded beta-sheet and double-stranded beta-sheet. Comparison with other classes of cystine knots provides indication that C-term VHv1.1 represents a new and distinct cystine knot motif. This analysis provides a structural basis for interpretation of the genetic and amino acid sequence data classifying polydnavirus gene products as members of cysteine-rich protein families.