Twists, Knots, and Rings in Proteins

STRUCTURAL DEFINITION OF THE CYCLOTIDE FRAMEWORK*,

K. Johan Rosengren, Norelle L. Daly, Manuel R. Plan, Clement Waine, and David J. CraikDagger

From the Institute for Molecular Bioscience, Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072, Australia

Received for publication, October 31, 2002, and in revised form, December 6, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Möbius cyclotides) and knots (a knotted arrangement of the disulfide bonds).

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cyclotides (1) are a remarkable family of small plant-derived proteins that have the unique feature of a circular protein backbone. They typically contain 28-37 amino acids linked in a continuous circle of peptide bonds and thus contain no free N or C terminus. They are further characterized by six conserved cysteine residues that form a tight network of disulfide bonds in their protein core. The cyclotides are extremely resistant to proteolysis and are remarkably stable. These features have led to suggestions that this protein family makes an excellent template for drug design applications (2). They also have exciting potential applications in agriculture because they are potent insecticidal agents (3).

The first reports of circular proteins in plants appeared in the mid-1990s, with the identification of viola peptide I, a hemolytic agent from Viola arvensis (4), the anti-HIV circulins from Chassalia parvifolia (5), the neurotensin antagonist cyclopsychotride A from Psychotria longipes (6), and kalata B1 from Oldenlandia affinis (7). Soon after, a series of peptides from viola plants was identified (1, 8), and the various peptides discovered to that point were recognized as part of a protein family that was named the cyclotides (1). Additional members have been discovered in the last few years (9-14). Table I shows a sequence alignment of the currently known cyclotides and identifies a number of conserved residues. These include the six cysteine residues as well as the highlighted residues in the six backbone loops between successive Cys residues. Loops 1 and 4 are particularly highly conserved.

                              
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Table I
Sequence alignment of peptides from the cyclotide family
The cyclotides can be subdivided into three families (bracelet, Möbius, and trypsin inhibitor). The conserved cysteine residues are boxed and numbered I-VI at the top of the sequence list. Other conserved residues are highlighted in color. The X in violapeptide I was not determined in the original report but is presumably Arg based on sequence homology.

Although its primary sequence and circular backbone were not characterized at the time, kalata B1 had been discovered in the 1970s as a bioactive peptide in a native medicine used by women in the Congo region of Africa to accelerate labor and childbirth. Gran (15) noted that the women ingested a tea made by boiling the aerial parts of O. affinis, and subsequent studies (16) suggested that an apparent 30-amino acid peptide isolated from the plant was an active uterotonic agent (17). The peptide was partially characterized at the time, but it was some 25 years later before it was established that the peptide contains 29 residues in a circular backbone (7).

Kalata B1 was the first cyclotide to be structurally characterized (7). It is a compact miniprotein that incorporates a distorted triple-stranded beta -sheet and several turns. Because of the resistance of the oxidized molecule to enzymatic cleavage it was not possible to determine its disulfide connectivity chemically, but NMR data suggested a I-IV, II-V, III-VI connectivity (7). This arrangement of disulfide bonds forms a cystine knot in which an embedded ring formed by the first two disulfide bonds and their connecting backbone segments is penetrated by the third (i.e. III-VI) disulfide bond. Such a cystine knot motif is now well known in a wide variety of proteins, ranging from growth factors to toxins, and occurs in a wide range of organisms (18, 19). However, the cyclotides remain as the only example in which a cystine knot is embedded within a circular protein backbone, a motif that is referred to as the cyclic cystine knot (CCK)1 (1, 20). A schematic representation of the conserved residues superimposed onto the cyclotide structural framework is given in Fig. 1.


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Fig. 1.   Schematic illustration of the cyclotide framework. The disulfide connectivities are shown by connections between the conserved cysteine residues and highlight the knotted arrangement. The position of the triple-stranded beta -sheet is indicated by arrows, with the arrow for the strand in loop 1, which is somewhat distorted, having jagged edges. The amino acids that are conserved throughout most of the cyclotide family are indicated by circles and single-letter amino acid codes. Those residues surrounded by white circles are present in the core of the molecule; those outside are highlighted with shaded circles. Structural roles for the conserved residues have been defined in the current study.

As is apparent from the sequences in Table I, most cyclotides fall into two families, referred to as the "Möbius" or "bracelet" cyclotides based on the presence or absence of a conceptual twist in the circular backbone resulting from a cis-peptide bond preceding a proline residue (1) in loop 5 of the sequence. The presence of this cis-Pro bond was tentatively suggested in the original report on the structure of kalata B1 (7) but was confirmed in subsequent reports (1) and is assumed to be present, based on sequence homology, in other members of the Möbius family.

A recent report (21) suggested an alternative possible disulfide connectivity for kalata B1 of I-VI, II-V, III-IV. This would have major topological implications because the structure would no longer be knotted, and indeed this connectivity represents a much simpler "laddered" arrangement of the disulfide bonds as indicated in Fig. 2. Clearly a more detailed structural analysis is required to resolve the ambiguity of the disulfide connectivity. In the current paper we show that a combination of NOE and coupling constant data defines the disulfide connectivity and cystine knot arrangement of prototypic cyclotides from both the Möbius and bracelet families.


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Fig. 2.   A schematic representation of the knotted disulfide connectivity originally proposed for kalata B1 is shown in A, and the laddered arrangement that has been suggested recently is shown in B. The laddered arrangement can unfold without breaking the bonds as indicated in C. The complexity of the knotted arrangement compared with the laddered is illustrated in the graph diagrams D and E, which show that although the knotted topology cannot be drawn in two dimensions without line-crossings, the laddered arrangement can.

Since the original structural reports many more cyclotides have been discovered. A major aim of the current study was thus to undertake a detailed structural analysis of the cyclotide framework to determine the structural significance of the conserved residues. Such an analysis is necessary to allow the exploitation of this framework as a template in drug design (2). In summary, in this paper we provide the first comprehensive analysis of the topological features in the unique family of cyclotide proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Möbius cyclotides), and knots (formed by conserved Cys residues of the family).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of Kalata B1 and Cycloviolacin O1-- Kalata B1 and cycloviolacin O1 were extracted from the aboveground parts of O. affinis and Viola odorata, respectively, with dichloromethane:methanol (1:1 v/v) overnight at room temperature. The extracts were partitioned with chloroform and water, and the water/methanol layer was concentrated on a rotary evaporator prior to lyophilization. The dried products were redissolved in H2O and purified on a preparative reverse phase C18 column (Vydac) at 8 ml/min. The molecular weights of kalata B1 (2,892.4) and cycloviolacin O1 (3,116.7) were confirmed by mass spectrometry to be identical to the expected values.

NMR Spectroscopy-- Samples for NMR spectroscopy were prepared by dissolving kalata B1 and cycloviolacin O1 in either 90% H2O and 10% D2O or 100% D2O to a final concentration of 5 mM and 1.3 mM, respectively. All spectra were recorded on Bruker ARX 500 and Bruker DMX 750 spectrometers with sample temperatures in the range 280-330 K. All spectra were acquired in phase-sensitive mode using time proportional phasing incrementation (22). For resonance assignment and structure determination a set of two-dimensional spectra including double quantum-filtered COSY (23), TOCSY (24) with a MLEV17 (25) isotropic mixing period of 80 ms, ECOSY (26) and NOESY (27) with mixing times of 100, 150, and 200 ms were recorded. The water proton signal in the COSY spectrum was suppressed by low power irradiation during the relaxation delay. For the TOCSY and NOESY spectra water suppression was achieved using a modified WATERGATE (water suppression by gradient-tailored excitation) (28) sequence. All two-dimensional spectra were collected over 4,096 data points in the f2 dimension and 512 or 600 increments in the f1 and processed using XWINNMR (Bruker). The f1 dimension was generally zero-filled to 2,048 real data points, with the f1 and f2 dimensions being multiplied by a sine-squared function shifted by 90° prior to Fourier transformation. Chemical shifts were internally referenced to sodium 2,2-dimethyl-2silapentane-5-sulfonate.

During the temperature titrations the temperature was increased in steps of 10 K from 280 to 330 K, and the chemical shift movements were documented by recording of one-dimensional and TOCSY spectra. Amide temperature coefficients were determined at pH 3.3 and at 5.8 for kalata B1 and at pH 2.8 and 5.3 for cycloviolacin O1. Similarly, the pH dependence was monitored for both kalata B1 and cycloviolacin O1 at 298 K by altering the sample pH from 1.6 to 6.5 by adding HCl and NaOH. The pKa of the titrating Glu3 residue was determined for both kalata B1 and cycloviolacin O1 by nonlinear curve fitting of the data points.

Structure Calculations-- For both peptides interproton distance restraints were derived from cross-peaks in NOESY spectra recorded with a mixing time of 200 ms. The cross-peaks were analyzed and integrated within the program XEASY (29) and assigned both manually and automatically by the NOAH function within the DYANA package (30). After an iterative process in which preliminary structures were used to resolve ambiguities, sets of 418 and 384 interresidual distance restraints, including 188 and 178 sequential, 88 and 98 medium, and 142 and 108 long range restraints, were derived for kalata B1 and cycloviolacin O1, respectively. In addition the spectral data allowed the introduction of 18 and 16 backbone dihedral angle restraints based on 3JHNHalpha coupling constants derived from the splitting of the amide signals in the one-dimensional spectra and 11 and 17 chi 1 dihedral angles based on 3JHalpha Hbeta coupling constants derived from ECOSY spectra together with NOE intensities derived from a 100-ms NOESY spectrum. Finally after analysis of preliminary structures and amide temperature coefficients 26 and 36 restraints for 13 and 18 hydrogen bonds were added to the structure calculations for kalata B1 and cycloviolacin O1, respectively. After initial structure calculations using DYANA (30), sets of 50 structures were calculated using a torsion angle simulated annealing protocol within CNS (31). This protocol involves a high temperature phase comprising 4,000 steps of 0.015 ps of torsion angle dynamics, a cooling phase with 4,000 steps of 0.015 ps of torsion angle dynamics during which the temperature is lowered to 0 K, and finally an energy minimization phase comprising 500 steps of Powell minimization. The resultant structures were subjected to further molecular dynamics and energy minimization in a water shell (32). The refinement in explicit water involved the following steps: 1) heating to 500 K via steps of 100 K, each comprising 50 steps of 0.005 ps of Cartesian dynamics; 2) 2,500 steps of 0.005 ps of Cartesian dynamics at 500 K before a cooling phase where the temperature was lowered in steps of 100 K, each comprising 2,500 steps of 0.005 ps of Cartesian dynamics; 3) minimizing the structures with 2,000 steps of Powell minimization. Identical protocols were used for both kalata B1 and cycloviolacin O1. Coordinates for both peptides have been deposited in the Protein Data Bank (kalata B1, 1NB1; cycloviolacin O1, 1NBJ).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Samples of kalata B1 and cycloviolacin O1 were isolated from O. affinis and V. odorata, respectively, and purified using our previously developed procedure (3). 750-MHz two-dimensional NMR spectra were recorded, assigned, and used to generate a series of distance, angle, and hydrogen-bonding restraints. These were in turn used to determine a high resolution structure of each peptide, chosen as representative examples from the Möbius and bracelet subfamilies of cyclotides. Of particular importance was a determination of the role of conserved residues in defining the extraordinarily stable CCK template and a definitive determination of the disulfide connectivity pattern. Previously, no structures of cyclotides have been solved at physiological pH, and the recent availability of a large number of cyclotide sequences has provided for the first time an opportunity to understand the fine structural details of the framework. We show here that the deprotonation of a conserved glutamic acid residue which occurs above pH 4 is crucial for stabilizing interactions in the CCK framework.

Disulfide Connectivity-- The six cysteine residues in kalata B1 were originally predicted to be most likely connected in a I-IV, II-V, III-VI pattern (7). However, based on an analysis of local intercysteine NOEs a recent study queried this interpretation and suggested that an alternative connectivity, i.e. I-VI, II-V, III-IV, was similarly possible, or indeed more likely (21). Therefore we sought additional evidence for the disulfide connectivity of kalata B1 and focused specifically on an analysis of the conformations of the cysteine side chains. Note that in this paper we use the numbering scheme starting at Cys I as specified in Table I, where Cys I-VI correspond to residues 1, 5, 10, 15, 17, and 22, respectively for kalata B1.

A key step in defining the disulfide connectivities in the current study was to determine the side chain dihedral angle chi 1 for the Cys residues by combining information from 3JHalpha Hbeta coupling constants with interproton HN-Hbeta distances (33). The former were extracted from ECOSY spectra, whereas the latter were derived from short mixing time NOESY spectra (see figure in supplemental material). For cysteines 1, 10, 15, and 22 a pattern of one large and one small 3JHalpha Hbeta coupling constant, together with one strong and one weak HN-Hbeta , NOE is observed, which is consistent with a chi 1 dihedral angle of -60°. For Cys5 two small 3JHalpha Hbeta coupling constants are observed, confirming a chi 1 dihedral angle of +60°, and Cys17 displays one large and one small 3JHalpha Hbeta coupling constant combined with two strong HN-Hbeta NOEs, consistent with a chi 1 dihedral angle of 180°. Such dihedral angle restraints were not used in either of the previous structure calculations for kalata B1, but examination of the structures shows that all six chi 1 values are consistent with the knotted topology, and only three are consistent with the "laddered" structure. Of these three, two are for the II-V bond, which is common to the two alternative connectivities (Fig. 2) and not in dispute.

To confirm further the connectivities we calculated a set of structures for each of the two suggested alternatives with chi 1 angle restraints explicitly included for the Cys residues. The resulting energies and restraints violations, which are summarized in Table II, clearly show that the inclusion of chi 1 dihedral restraints provides the key information to confirm that the knotted arrangement is favored over the laddered form.

                              
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Table II
Energy and structural statistics for the families of 20 structures representing the solution structures of cycloviolacin O1 and kalata B1

Temperature and pH Titration-- Previous studies of cyclotide structures (1, 7, 34, 35) have all been done at pH <4, as is typically the case for NMR-derived structures of small proteins because amide exchange rates increase at higher pH values and can lead to broadening and/or loss of signals. In the current study it was of interest to determine high resolution structures at closer to physiological pH and to monitor potential effects of pH on structure. In addition, we had noted earlier an unusually large temperature dependence of signals from Asn11 in kalata B1. Because the cyclotide framework contains an extensive network of hydrogen bonds it was of interest to determine the origin of this anomalous effect.

Fig. 3 shows the chemical shift as a function of pH for the amide protons in kalata B1 and cycloviolacin O1. As expected, most backbone amides vary by less than 0.1 ppm in chemical shift over the pH range 2-7, showing that the global fold is generally unaffected by pH. However, a surprisingly large effect is seen for two or three residues in each molecule. Both kalata B1 and cycloviolacin O1 contain only one glutamic acid residue, and monitoring the pH dependence of the gamma  protons or other nearby protons allows calculation of its pKa value. An apparent pKa for Glu3 of 3.5 for kalata B1 and 3.0 for cycloviolacin O1 was calculated from this analysis. The value for kalata B1 is similar to that reported recently from an analysis of 15N heteronuclear single quantum coherence spectra (21). It is clear from the pH titrations that the protonation/deprotonation of Glu3 significantly affects the chemical shifts of the backbone amide protons of residues 11 and 12 in kalata B1 and residues 11, 12, and 13 in cycloviolacin O1. For cycloviolacin O1 the amide shifts and the Halpha shifts of most other residues in loop 3 are also affected, indicating a local conformational change upon protonation of Glu3. It is interesting to note that with the sole exception of the Halpha of Thr11, the chemical shifts of all Halpha protons move toward their random coil shifts when the pH is lowered, suggesting that loop 3 becomes less structured. Further, at pH values above the pKa of Glu3 we observe additional NOE connectivities between the Glu3 side chain protons and loop 3 amide protons in both peptides, which further supports the presence of a local conformational change.


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Fig. 3.   pH titration monitoring amide proton chemical shifts in kalata B1 (left) and cycloviolacin O1 (right). Proton chemical shifts are represented as a function of pH for all protons displaying a pH dependence of more than 0.1 ppm in the investigated pH range of 1.6-7.0. Although cycloviolacin O1 has a larger number of amide shifts exceeding this threshold, most are only slightly larger. A substantial pH dependence is observed only for Asn11 and Thr12 in kalata B1, and Thr11, Val12, Thr13 in cycloviolacin O1.

Amide temperature coefficients are useful for indicating the presence of hydrogen bonds (36). For cycloviolacin O1 these data suggest the presence of strengthened hydrogen bonding interactions involving the Glu3 carboxyl group in the deprotonated state, consistent with the conformational change observed with pH. In particular, on deprotonation of Glu3, the amide temperature coefficient of all of the amide protons in loop 3 of cycloviolacin O1 are lowered in magnitude by an average of 1 ppb/K. Hence, it was of interest to determine high resolution structures at a pH (~6) where Glu3 is deprotonated, as described below.

Structure Determination-- Solution structures were determined for both kalata B1 and cycloviolacin O1 by simulated annealing using experimental distance restraints based on NOESY cross-peaks and dihedral angle restraints based on coupling constants. Particular emphasis was placed on obtaining ultrahigh resolution structures by careful refinement in a water box. Of the final 50 structures for each peptide families of the 20 lowest energy structures consistent with experimental data were chosen to represent the structures of kalata B1 and cycloviolacin O1. A summary of the energetic and geometric statistics for these families is given in Table II. The structures are in excellent agreement with the experimental data, showing no distance violation greater than 0.2 Å and no dihedral angle violation greater than 2°. As indicated by the overlay of the structures in Fig. 4, A and B, it is clear that the peptides have well defined structures. Indeed the structural families not only give information on the backbone fold but also provide detailed information on the positioning of the side chains.


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Fig. 4.   Solution structures of kalata B1 and cycloviolacin O1. Kalata B1 (A) and cycloviolacin O1 (B) are shown as stereoviews of the 20 lowest energy structures of kalata B1 and cycloviolacin O1 superimposed over the backbone atoms. C and D show the lowest energy structures for kalata B1 and cycloviolacin O1, respectively, with elements of regular secondary structure represented in ribbon style and the cysteine side chains as balls and sticks.

The general fold of the cyclotides can be described as an elongated peptide circle that folds back on top of itself and is stabilized by three cross-bracing disulfide bonds, which link diagonally opposed beta -strands. As shown in Fig. 4, C and D, the main element of secondary structure is an antiparallel beta -sheet. Strand 1 (residues 14-18 in kalata B1 and 19-21 in cycloviolacin O1) and strand 2 (residues 21-25 in kalata B1 and 24-26 in cycloviolacin O1) are linked by a beta -turn, hence forming a traditional beta -hairpin motif. A third strand, which incorporates residues 3-5 in both kalata B1 and cycloviolacin O1, is more loosely associated with the sheet and not formally recognized as a regular beta -strand by PROMOTIF (37). In addition to the central beta -sheet the cyclotide structures comprise a number of well ordered tight turns. In kalata B1 four beta -turns, including a type I (residues 5-8), a type II (residues 12-15), a type VIa1 (residues 18-21), and a type I' (residues 23-26) are present, whereas cycloviolacin O1 incorporates a type I beta -turn (residues 5-8), a type I' beta -turn (residues 21-24), and a classical gamma -turn (residues 28-30). In addition, the longer loop 3 in cycloviolacin O1, which involves residues 12-18, adopts a 310 helical conformation. These structural elements are all stabilized by an extensive network of hydrogen bonds and hydrophobic interactions, and together with the disulfide bonds they account for the exceptional stability of the cyclotides.

Structural Roles of Individual Amino Acids in the Cyclotide Framework-- The high resolution structures obtained in this study provide an opportunity to determine the roles of the individual amino acids in defining and stabilizing the structures of kalata B1 and cycloviolacin O1 and more broadly for defining their importance for the CCK motif. We therefore undertook a detailed analysis of the geometries, hydrogen bonding interactions, and charge distributions in the two structures. The results are summarized in Table III.

                              
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Table III
Structural roles of individual amino acids in the cyclotides based on the structures of kalata B1 and cycloviolacin O1

From this analysis the reasons for the conservation of certain residues are clear, with most of the highly conserved residues having prominent structural roles in the cyclotide framework. One example of this is the conservation of glycine residues. Glycine is the only amino acid for which a negative phi -angle conformation is not specifically favored and therefore is often seen in tight turns where adaptability of the backbone geometry is important. That this role is important for the cyclotides is illustrated by the fact that four of five glycines in kalata B1 and both of the two glycines in cycloviolacin O1 adopt positive phi -angles.

A second example concerns Glu3, which, apart from the six cysteines, is the only residue fully conserved throughout both the Möbius and the bracelet families. From the structures it is clear that the side chain oxygens of the glutamic acid act as acceptors for multiple hydrogen bonds linking loops 1 and 3. For cycloviolacin O1 it is clear that by accepting hydrogen bonds from the backbone amides of residues 11, 12, and 13 at high pH Glu3 plays an important role in stabilizing the short 310 helix. In addition, at high pH the Thr13 hydroxyl proton, which is normally not visible because of fast exchange with the solvent, appears in the spectra, indicating that the presence of an additional side chain-side chain hydrogen bond has reduced the exchange rate of this proton. Similarly, from an analysis of the kalata B1 structure it is clear that even though there is no helix present Glu3 still interacts closely with loop 3. The backbone amide protons of Asn11 and Thr12 and the side chain hydroxyl of Thr12 are all within the expected distance and geometry for hydrogen bonding interactions with the two oxygen atoms in the Glu3 side chain. This is consistent with the findings of Skjeldal et al. (21), who reported the possibility of hydrogen bonds to Glu3 but did not identify specific interactions.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

When the structure of kalata B1 was first reported in 1995 (7) there were only a few examples of macrocyclic peptides known and at that stage it was not recognized that they would be part of a large family. With the wealth of sequences now reported there is an opportunity to correlate structural elements in prototypic cyclotides with conserved sequence elements across a family of nearly 50 peptides to gain an understanding of the role of individual residues in structure and function. Before addressing these broad structural issues it is important to address definitively the issue of the disulfide connectivity and to comment further on the presence of a cis-peptide bond, i.e. to define the knots and twists characteristic of the cyclotides.

Cystine Knot or Not?-- In small disulfide-rich proteins the cysteine residues are often packed tightly together in the protein core, and determination of the disulfide connectivity can be a significant challenge (38). That this is especially true for kalata B1 has been highlighted by the fact that two independent reports have predicted different connectivities for the three disulfide bonds. In previous studies by Saether et al. (7) and by Skjeldal et al. (21), distances were derived from NMR data and subjected to simulated annealing in attempts to predict which disulfide connectivity is most consistent with the experimental data. In addition, a limited number of backbone dihedral restraints were used in the study by Saether et al. A striking result from these studies is that many of the theoretically possible disulfide connectivities are perfectly consistent with the constraint data. In other words, the use of even a large amount of NOE distance data to define the global fold accurately does not necessarily discriminate between possible disulfide connectivities.

Given the ambiguity associated with the global fold approach to determine disulfide connectivities, can local intercysteine NOEs be used diagnostically instead? For the cyclotides the current paper shows that the answer is clearly no. Skjeldal et al. (21) analyzed the NOE data for intercysteine Hbeta -Hbeta connectivities. On the basis that Hbeta -Hbeta distances between two covalently linked cysteines is well within the range observed in NOESY spectra this approach could in principle provide clues toward the disulfide connectivity (39), but for molecules like kalata B1, where all of the cysteine residues are packed closely together, it can be misleading because of the proximity of nonbonded and bonded cysteines. Fig. 5 shows an analysis of intercysteine distances for kalata B1 and highlights several examples of Hbeta cysteine protons that are as close, or even closer to cysteine protons involved in neighboring disulfide bonds than to protons from the covalently bound partner.


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Fig. 5.   Conformations of the cysteine side chains in the cystine knot in kalata B1. A shows selected interproton distances between connected (solid arrows) and not connected (dotted arrows) cysteine beta -protons. It is clear that distances between nonconnected cysteines can be very close, thus highlighting the difficulty of using NOE data to determine disulfide connectivities. The distances measured from the structure of kalata B1 are given in Å units. B shows chi 1 dihedral angles as determined experimentally by NOE and coupling constant analysis. From this it is clear that by locking the cysteine side chains in the given orientation the alternative pairing of Cys1-Cys22 and Cys10-Cys15 is not feasible.

To determine definitively the disulfide connectivity, in the present study we focused on the chi 1 dihedral angles of the cysteine residues. The rationale behind this was that although it is possible for one backbone fold to accommodate more than one possible pairing of the cysteine residues, these alternatives require different cysteine side chain orientations around the Calpha -Cbeta bond. Our results provide clear experimental evidence for fixed side chain conformations of all six cysteine side chains in kalata B1 (Fig. 5B). Strikingly, the results show that the observed chi 1 angles for the cysteines are all consistent with the I-IV, II-V, III-VI cysteine pairing, giving kalata B1 a knotted topology as originally proposed by Saether et al. (7). By contrast, for the nonknotted topology suggested by Skjeldal et al. (21) only three of six chi 1 angles agree with the experimental data. Not surprisingly, two of these are Cys5 and Cys17, which are connected in both suggested pairings. Further, we have shown that the introduction of dihedral angle restraints that force the cysteine side chains to adopt the experimentally observed local conformation results in calculated structures perfectly consistent with all other constraint data. These results are definitive evidence for the I-IV, II-V, III-VI pairing of the six cysteines, reinforcing the importance of the major characteristic of the cyclotides, the CCK motif.

It is also relevant to note that the exceptional stability of the cyclotides is perfectly consistent with a knotted and braced arrangement of disulfide bonds, whereas a laddered arrangement would not be expected to be so stable. The laddered topology is present in another family of small disulfide-rich peptides known as the theta -defensins (40, 41). In that case the structures display a much greater degree of flexibility than is seen here for the cyclotides (42).

A Backbone Twist Caused by a cis-Peptide Bond Defines the Möbius Subfamily of Cyclotides-- The kalata B1 sequence incorporates three proline residues. Strong sequential Halpha -Hdelta NOE cross-peaks for Thr12-Pro13 and Leu27-Pro28 confirmed trans-peptide bonds, whereas a strong sequential Halpha -Halpha cross-peak between Trp19 and Pro20 confirmed a cis-peptide bond, consistent with what has been reported by Skjeldal et al. (21). In the initial report on the structure of kalata B1 we noted potential ambiguity on the cis/trans conformation of Pro20 but subsequently confirmed the cis arrangement and proposed the naming scheme "Möbius cyclotides" for peptides like kalata B1 which contain this twist in the circular backbone (1). Cycloviolacin O1 incorporates two proline residues, Pro9 and Pro30, and for both a trans-peptide bond conformation was confirmed by NOE cross-peaks, making it a member of the bracelet cyclotides, which contain an all-trans-circular backbone. The strong sequence conservation within the subfamilies of cyclotides shown in Table I and the presence/absence of a cis-backbone twist in the Möbius/bracelet prototypic cyclotides studied here reinforce the basis of this naming scheme.

Topological Features and Structural Role of Conserved Residues in the Cyclotide Framework-- The structural role of individual residues in the cyclotides is summarized in Table III. When analyzing the sequences in conjunction with the structures it is apparent that although only a limited number of structural features appear to be absolutely essential to maintain the overall fold, several others that are conserved in most cyclotides provide additional stability. Fig. 1 shows a superimposition of the key residues involved in these interactions on a schematic representation of the CCK framework. An immediate observation is that the core region that includes the cystine knot is highly conserved. In particular, it is interesting to note that loop 1 is the most conserved among currently known cyclotides and that apart from the six cysteine residues, Glu3 is the only residue fully conserved throughout all Möbius and bracelet cyclotides. Furthermore, only very conservative changes are observed for the other residues in this loop, i.e. Ala/Gly or Ser/Thr. Other conserved features include hydrophobic residues at positions corresponding to Val6 and Val21 in kalata B1 in addition to a hydroxyl-containing residue in loop 3. Finally there is a high degree of conservation in loop 4 and of several residues in loop 6.

The lack of sequence variation of loops 1 and 4 suggests that the core of the cyclotides is structurally optimal and not amenable to significant changes. Comparison of the cystine knot of kalata B1 and cycloviolacin O1 shows that not only are the residues highly conserved, but the backbone and side chain angles are as well, as indicated by Fig. 6, A and B. In kalata B1 Gly2 adopts a phi -angle of ~60°. However, in cycloviolacin O1, which is one of only two cyclotides with the Gly substituted for an Ala at this position, a positive phi -angle is not an absolute necessity, and the Ala adopts a phi -angle of ~ -120°. This angle and the psi -angle of Cys1 are the only angles that differ between the cystine knot of kalata B1 and cycloviolacin O1. It is interesting that despite the extremely high conservation in the CCK of the cyclotides the cystine knot motif itself is not restricted to these residues. Significant sequence and loop size variation is observed across other known cystine knot proteins (19, 43).


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Fig. 6.   Comparison of structural features of kalata B1 (A, C, E, and G) and cycloviolacin O1 (B, D, F, and H). The geometry of the embedded ring of the cystine knot, which is made up by backbone loop 1 (comprising residues Cys1-Cys5) and loop 4 (comprising residues Cys15-Cys17) and their connecting disulfide bonds, is highly conserved, with the only difference being a rotation of the Cys1-Ala2 peptide bond, as indicated by the curved arrows in A and B. Loop 3 in both folds is stabilized by hydrogen bonding to the Glu3 side chain as shown in C and D, where conserved hydrogen bonds are indicated by broken lines. These hydrogen bonds are clearly important for the structure, which in kalata B1 involves a tight turn, and in cycloviolacin O1 the backbone folds into a short 310 helical arrangement. E and F show that though the turn arrangements are different, loops 2 and 5 interact closely both in kalata B1 and cycloviolacin O1. The presence of a number of hydrophobic contacts is highlighted by the ellipses. Loop 6, as indicated by G and H, is characterized by hydrophobic interactions. The Tyr26 side chain in cycloviolacin O1 is able to adopt two conformations as evident both from structure calculations and experimental data and indicated by a curved arrow in H. The two conformations are stabilized by hydrophobic interactions with Pro30 and hydrogen bonding to Gly28, respectively.

It is not unexpected that the cystine knot, which is the main characteristic of the cyclotides, plays a major role in stabilizing the overall fold. In contrast, the importance of Glu3 for linking loop 3 to the protein core was not obvious from analysis of the sequences alone. The significance of this residue was highlighted initially by pH and temperature titrations and confirmed by structural analysis. From an analysis of the kalata B1 structure it is clear that Glu3 interacts closely with loop 3 (Fig. 6C). Three hydrogen bond donors are within the expected distance and geometry for interacting with the two oxygen atoms in the Glu3 side chain. These are the backbone amides of Asn11 and Thr12 and the side chain hydroxyl of Thr12. For cycloviolacin O1 the Glu3 carboxylate group is part of a unique arrangement in which both oxygen atoms coordinate two hydrogen bonds each and thereby form a cap at the N terminus of the helix in loop 3, as shown in Fig. 6D. The side chain hydroxyl of Thr13 is involved in this network of hydrogen bonds, and the fact that a hydroxyl group in the form of a Ser or Thr is present at this position in all cyclotides suggests that this latter interaction is conserved throughout the entire cyclotide family. Further evidence for the importance of the interactions between loop 1 and loop 3 both in kalata B1 and cycloviolacin O1 is given by the low pKa values (3.5 and 3.0, respectively) observed for Glu3, indicating that the deprotonated state is stabilized by favorable interactions.

Another structurally conserved element of the cyclotides is an antiparallel beta -sheet comprising a beta -hairpin associated with a distorted third strand. The beta -hairpin spans loops 4 and 5 and includes the first residue of loop 6, with loop 5 comprising the turn region. Sequence analysis (Table I) suggests that conservation of the turn residues is not critical for formation of the beta -hairpin. Elements of the beta -hairpin also appear to be able to form in the absence of the turn residues, based on a previous study on acyclic permutants of kalata B1 (44). In addition to the backbone hydrogen bonds observed between beta -strands there is a network of side chain hydroxyl group hydrogen bonds across the hairpin which provide additional stability. In kalata B1 these include Thr16-Thr23, Thr23-Ser18, and Ser18-Cys17. Similarly in cycloviolacin O1 hydrogen bonds appear to be present between Ser19-Tyr26 and Ser21-Cys20. Additional stabilization is provided by the conserved cysteines, of which two cross-link strand 1 in the beta -hairpin to strand 3, which comprises loop 1. Further, there are a number of hydrophobic interactions between loops 2 and 5, which may be involved in stabilizing this motif. In both Möbius and bracelet cyclotides there is a strong preference for a hydrophobic residue at the start of loop 2 (position 6), with the only exceptions being palicourein, kalata B7, and varv peptide F, which all have a hydrophobic residue at position 7 instead. The importance of this is clear from the structural analysis, which reveals a number of contacts between this hydrophobic residue (generally Val) and Trp19 and Pro20 in kalata B1, as shown in Fig. 6E. Also, in cycloviolacin O1 the Val forms part of a hydrophobic patch by interacting mainly with Tyr7 and the hydrophobic region of the Arg23 side chain (Fig. 6F).

Of the loops that are not associated with the core, loop 6 is the most conserved. As shown in Fig. 6, G and H, the main structural characteristic of this loop is the clustering of hydrophobic side chains. The sequence conservation observed in loop 6 may be a result of its putative involvement in processing reactions of mature cyclotides from their precursor proteins (3). At this stage little is known about the processes involved in cleavage and cyclization; however, they are likely to be catalyzed by enzymes. A high degree of conservation is likely to be required for interaction with enzymes or indeed for alternative nonenzymatic reactions such as is seen with inteins.

In general, peptides and proteins tend to have their hydrophobic side chains packed tightly together in a hydrophobic core. However, in the cyclotides the core is almost completely filled by the cysteines, resulting in many hydrophobic groups being solvent exposed. This is shown in Fig. 7, which illustrates the surface properties of kalata B1 and cycloviolacin O1. The main hydrophobic patch involves regions of loops 2, 5, and 6. In kalata B1 the side chain of Val21 forms the center of the patch by interacting with Val6, Pro20, Pro28, and the methyl of Thr4. The importance of this clustering is obvious from the conservation of a hydrophobic residue at this position. The sole exception is the bracelet member cycloviolin B, in which a Gln is found at position 21 (i.e. the final residue of loop 5). However, cycloviolin B is also the only cyclotide that lacks the Pro corresponding to position 28 in kalata B1 and thus has an overall reduced hydrophobic nature.


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Fig. 7.   Nature of the protein surface of representative cyclotides. A and B show the orientation used to view the surfaces of kalata B1 (C) and cycloviolacin O1 (D), respectively. The surface of individual residues is colored based on their properties, with green, blue, yellow, white, red, and purple representing hydrophobic, glycines, cysteines, hydrophilic, negative, and positive residues, respectively. From C and D it is clear that a major hydrophobic patch involving loops 2, 5, and 6 is present in kalata B1 and cycloviolacin O1. In contrast, on the other face of the molecules (shown in E and F, respectively, for kalata B1 and cycloviolacin O1), there are clearly differences in the surface nature, with cycloviolacin incorporating an additional hydrophobic patch because of the hydrophobic nature of the extended loop 3. E and F are rotated 180° in relation to C and D.

Concluding Remarks-- The high affinity and selectivity of many peptides for physiologically important receptors suggest that they make excellent drug leads. However, the use of peptides in pharmaceutical applications is limited by their poor stability and bioavailability. The cyclotide framework is an extremely stable and compact structure. To achieve such stability in a small framework it has evolved a highly conserved set of structurally important residues. In addition to the six Cys residues that make up the knotted core, a crucial Glu residue is involved in key hydrogen bonding interactions. The core features also include a beta -sheet, which is stabilized by a number of main chain and side chain hydrogen bonds as well as hydrophobic clusters. Onto this remarkable framework several "loop cassettes" have evolved. Although these cassettes form well ordered stable turns, or in the case of loop 3 in cycloviolacin O1, a short 310 helix, they do not have major roles in stabilizing the overall fold. This ability of this remarkable framework to accommodate a variety of different loops suggests that it is highly suited for grafting of bioactive epitopes and indeed provides a unique opportunity for the design of novel drugs.

    ACKNOWLEDGEMENT

We thank Ulf Göransson for helpful comments.

    FOOTNOTES

* This work was supported in part by a grant from the Australian Research Council (to D. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a figure and legend.

The atomic coordinates and the structure factors (code 1NB1 (kalata B1) and 1NBJ (cycloviolacin O1)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger Australian Research Council senior fellow. To whom correspondence should be addressed. Tel.: 61-7-3365-4945; Fax: 61-7-3365-2487; E-mail: d.craik@imb.uq.edu.au.

Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211147200

    ABBREVIATIONS

The abbreviations used are: CCK, cyclic cystine knot; COSY, correlation spectroscopy; ECOSY, exclusive correlation spectroscopy; NOE, nuclear Overhauser effect; TOCSY, total correlation spectroscopy; NOESY, NOE spectroscopy.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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