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The cystine knot structure of ion channel toxins and related polypeptides
PII: S0041-0101(98)00149-4
Toxicon Vol. 36, No. 11, pp. 1573±1583, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0041-0101/98 $19.00 + 0.00
THE CYSTINE KNOT STRUCTURE OF ION CHANNEL TOXINS AND RELATED POLYPEPTIDES RAYMOND S. NORTON* and PAUL K. PALLAGHY Biomolecular Research Institute, 343 Royal Parade, Parkville, 3052, Australia (Received 12 February 1998; accepted 10 April 1998)
R. S. Norton and P. K. Pallaghy. The cystine knot structure of ion channel toxins and related polypeptides. Toxicon 36, 1573±1583, 1998.ÐAn increasing number of ion channel toxins and related polypeptides have been found to adopt a common structural motif designated the inhibitor cystine knot motif (Pallaghy P. K., Nielsen, K. J., Craik, D. J., Norton, R. S. (1994) A common structural motif incorporating a cystine knot and triple-stranded bsheet in toxic and inhibitory polypeptides. Protein Science 3, 1833±1839). These globular, disul®de-stabilized molecules come from phylogenetically diverse sources, including spiders, cone shells, plants and fungi, and have various functions, although many target voltage-gated ion-channels. The common motif consists of a cystine knot and a triple-stranded, anti-parallel b-sheet. Examples of ion-channel toxins known to adopt this structure are the o-conotoxins and o-agatoxins, and, more recently, robustoxin, versutoxin and protein 5 from spiders, as well as k-conotoxin PVIIA and conotoxin GS from cone shells. The variations on the motif structure exempli®ed by these structures are described here. We also consider the sequences of several polypeptides that might adopt this fold, including SNX-325 from a spider, d-conotoxin PVIA and the mO-conotoxins from cone shells, and various plant and fungal polypeptides. The interesting case of the two- and three-disul®de bridged binding domains of the cellobiohydrolases from the fungus Trichoderma reesei is also discussed. The compact and robust nature of this motif makes it an excellent scaold for the design and engineering of novel polypeptides with enhanced activity against existing targets, or with activity against novel targets. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
It has been suggested that the number of protein families (that is, groups of proteins with recognizable sequence similarity) to be found in nature is about 1000, and that the number of unique protein folds is even lower (Brenner et al., 1997). Among small, disulfide crosslinked polypeptides, several different folds have been recognized. One that is found in an increasing number of small toxic and inhibitory polypeptides with diverse functions and origins is the inhibitor cystine knot (ICK) structural motif (Narasimhan et al., 1994; * Author to whom correspondence should be addressed. Email: [email protected] 1573
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Pallaghy et al., 1994). Polypeptides that adopt this structure have a range of interesting biological activities, several of which are relevant to the development of new human therapeutics. They are also of interest as scaffolds for protein engineering and phage display. In this article, we describe several recently-determined structures that share this motif, compare the motif with structural motifs found in other disulfide cross-linked polypeptides, and discuss additional polypeptide sequences that would be expected to adopt this motif structure. The ICK structural motif is characterised by a triple-stranded, anti-parallel b-sheet stabilized by a cystine knot. In terms of amino acid sequence, all known examples of this fold satisfy the following consensus: CX3±7CX3±6CX0±5CX1±4CX4±13C, where X can be any amino acid. The ICK motif is distinct from the growth factor cystine knot motif, found in transforming growth factor-b2, platelet-derived growth factor and nerve growth factor (McDonald and Hendrickson, 1993; Isaacs, 1995). It includes several plant protease inhibitors described as knottins (Le Nguyen et al., 1990), but expands the definition beyond these proteins and provides a more representative description of the motif. Structural motifs are known to occur in molecules with different functions and from diverse sources (Orengo et al., 1993). However, in the case of the ICK motif, the similarity goes beyond a simple topological similarity as it also involves the half-cystine residues occurring in fixed positions relative to the b-sheet hydrogen bonding pattern. This is reminiscent of sequence motifs such as the zinc finger motif (Berg, 1988), except that these motifs are predictive for a specific function and the sequence examples retain some homology among non-cystine residues. In the ICK motif this is not the case, and the functions are quite diverse. The fold, presumably dictated largely by the gaps between the half-cystine residues and their pairing pattern, appears to be an ideal compact globular scaffold for the presentation of a variety of functional groups, thereby generating a range of polypeptides with diverse biological targets. An interesting protein engineering application has already been demonstrated in the case of a chimera of a trypsin inhibitor and a carboxypeptidase inhibitor that retains both functions (see below). Exploitation of the ICK motif by phage display technology promises to create new biological activities with high potency. SEQUENCES
Figure 1 presents a non-redundant sequence alignment of representative members of the inhibitor cystine knot motif with distinct molecular targets and experimentally determined tertiary structures. These sequences come from organisms as diverse as spiders, coneshells, plants and fungi. As indicated above, they all satisfy the consensus sequence CX3ÿ7 CX3ÿ6 CX0ÿ5 CX1ÿ4 CX4ÿ13 C where X can be any amino acid (including half-cystine). An earlier version of this description, using the consensus sequence CX3±7CX4±6CX0±5CX1±4CX4±10C, enabled a search of amino acid sequence data bases to identify three polypeptides from diverse sources that should adopt this fold (Pallaghy et al., 1994). These were gurmarin, a sweet taste suppressor from the tree Gymnema sylvestre, huwentoxin-I, from the Chinese birdeating spider Selenocosmia huwena, and the race-specific elicitor AVR9, from the fungal tomato pathogen Cladosporium fulvum. Solution structures have since been reported for all three of these (Arai et al., 1995; Qu et al., 1997; Vervoort et al., 1997), and they confirm the predicted fold. A number of hits were also found in larger proteins but these were not
Fig. 1. Sequence alignment of members of the ICK motif for which experimentally determined structures are available and which have non-redundant sequences or distinct functions. The half-cystine residues of the motif are highlighted in bold. The continuous lines represent the motif disul®de bridges and the dashed lines the additional bridges. The arrows represent the typical motif b-strands. The SWISS-PROT or PIR database accession codes (the latter indicated by `PIR') and literature references are given for the amino acid sequences of all polypeptides shown here, followed by the Brookhaven Protein Data Bank (Bernstein et al., 1977) accession codes (where available), and literature references for the structures, as follows: robustoxin from Atrax robustus (TXRO_ATRRO, Sheumack et al., 1985; 1qdp, Pallaghy et al., 1997), o-ACTX-Hv1 from Hadronyche versuta (TXO6_HADVE; 1axh, Fletcher et al., 1997b), o-Aga IVB from Agelenopsis aperta (TX4B_AGEAP, Adams et al., 1993; 1oma, Yu et al., 1993), m-Aga I from Agelenopsis aperta (TXM1_AGEAP, Skinner et al., 1989; 1eit, Omecinsky et al., 1996), huwentoxin-I from Selenocosmia huwena (PIR-A37479, Liang et al., 1993; Qu et al., 1997), protein 5 from Brachypelma smithii (TXP5_BRASM, Kaiser et al., 1994; Hill et al., unpublished), o-CgTx GVIA from Conus geographus (CXO6_CONGE, Olivera et al., 1984; 2CCO, Pallaghy, and Norton, submitted), o-CmTx MVIIC from Conus magus (CXOC_CONMA, Hillyard et al., 1992; 1omn, Farr-Jones et al., 1995), k-CpTx PVIIA from Conus purpurascens (Terlau et al., 1996; 1av3, Scanlon et al., 1997), conotoxin GS from Conus geographus (CXGS_CONGE, Yanagawa et al., 1988; 1ag7, Hill et al., 1997), CMTI-I from Cucurbita maxima (ITR1_CUCMA, Wilusz et al., 1983; 3cti, Nilges et al., 1991), CPI from Solanum tuberosum (MCPI_SOLTU, Hass et al., 1975; 4cpa, Rees and Lipscomb, 1982), gurmarin from Gymnema sylvestre (PIR-JX0200, Kamei et al., 1992; 1gur, Arai et al., 1995), kalata B1 from Oldenlandia anis (PIR-A56283, Saether et al., 1995; 1kal, Saether et al., 1995), and AVR9 from Cladosporium fulvum (AVR9_CLAFU, Van Kan et al., 1991; Vervoort et al., 1997).
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considered further, usually because these proteins contained several additional halfcystines and it was not clear that the half-cystines which satisfied the motif consensus were linked to one another. However, as discussed below for cellobiohydrolase, it is certainly possible for small domains of large proteins to adopt the motif structure. The third and fourth half-cystines of the motif are usually contiguous, and always so for spider and cone-shell polypeptides. Gurmarin, however, is the only currently known representative from the plant kingdom that satisfies this criterion. Interestingly, the fungal polypeptide AVR9, which induces defence responses in plants resistant to the producing fungus, resembles the plant protease inhibitors and kalata in having a finite number of residues between the third and fourth half-cystines, although the functional significance of this is unknown at present. Most of the members have only the three disulfide bridges that comprise the cystine knot, although there are three (nonredundant) examples that contain a fourth disulfide bridge: robustoxin (otherwise known as d-ACTX-Hr1), o-agatoxin IVB (o-Aga-IVB) and m-agatoxin I (m-Aga-I). Robustoxin has an unusual disulfide pairing pattern, characterised in the sequence by a triplet of half-cystine residues, the first two being the third and fourth of the cystine knot motif. The additional disulfide bridge links the loop between the fourth and fifth half-cystine residues to a C-terminal extension of the sequence. o-Aga-IVB and m-Aga-I have an additional disulfide bridge that stabilizes the fifth loop and is located between the fifth and sixth half-cystine residues of the motif. Several amino acid sequences have been reported recently that either satisfy or closely resemble the consensus sequence. d-conotoxin PVIA, from the fish-hunting cone shell Conus purpurascens, has novel `lockjaw' activity in fish due to its retardation of sodium channel inactivation (Shon et al., 1994; Terlau et al., 1996) and satisfies the consensus sequence. The mO-conotoxins MrVIA and MrVIB, from the cone shell Conus marmoreus, are sodium channel and L-type calcium channel inhibitors (Fainzilber et al., 1995; McIntosh et al., 1995). If they adopt the motif structure, this would require an expansion of the second gap size, from X3±6 to X3±9. SNX-325, from the spider Segestria florentina, probably inhibits N-type calcium channels (Newcomb et al., 1995) and would require a major revision of the last gap size (from X4±13 to X4±20). If confirmed as a new member of the motif, it would contain a larger, but otherwise similar, disulfide-stabilized fifth loop, as found in o-Aga-IVB and m-Aga-I. The existing member of the family with the largest last gap (13 residues) is o-ACTX-Hv1 which, however, has no additional disulfide bridge. The insect a-amylase inhibitor from the Mexican crop plant Amaranthus hypocondriacus satisfies the consensus sequence and was in fact homology modelled using a trypsin inhibitor from Ecballium elaterium (highly homologous to CMT-I in Fig. 1) and the binding domain of cellobiohydrolase I (see below) (Chagolla-Lopez et al., 1994). The chitin binding polypeptides, AMP1 and AMP2, from the plant Mirabilis jalapa, inhibit the growth of plant pathogenic fungi (Broekaert et al., 1992; Cammue et al., 1992). If they were shown to be members of the ICK motif structural family, the second gap size would have to be expanded from X3±6 to X3±8, and the motif would then be endowed with members from plants that attack fungi and vice-versa. The naming of the motif (Pallaghy et al., 1994) continues to be appropriate in terms of the known functions of the majority of polypeptides in this structural family. Both the spider and cone-shell sequences include blockers for the three major voltage-gated ion channel types (Na+, K+ and Ca2+). Neither of the plant kingdom members with known molecular targets is an ion-channel blocker; rather, both are protease inhibitors. The putative plant and fungal members of the motif include other enzyme inhibitors and binding polypeptides. Some new members of the family, for example robustoxin, modulate
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rather than block the function of their target receptor, in this case the voltage-gated Na+ channel, but as a general descriptor the name is still adequate. STRUCTURES
The motif structure (Fig. 2) consists of an anti-parallel, triple-stranded b-sheet stabilized by a cystine knot (Narasimhan et al., 1994; Pallaghy et al., 1994). The cystine knot is comprised of a ring formed by two disulfide bridges and the interconnecting backbone, through which the third disulfide passes. The first b-strand of the motif is typically three residues in length with the third position being a half-cystine residue, although the strand can be highly distorted or linked to the sheet by only a single b-bridge, as in robustoxin, oACTX-Hv1 and conotoxin GS. The later two b-strands form a hairpin structure, with the strand lengths typically being three and four residues, respectively. Half-cystine residues fill positions two and three, respectively, of b-strands II and III. The defining characteristics of the motif up to this point (Pallaghy et al., 1994) are that these later two half-cystine residues pack against each other in the b-sheet, and the half-cystine of bstrand III (being the central strand) packs against the first residue of b-strand I (two residues preceding the half-cystine of b-strand I). The ICK motif topology is babb-like, with the a segment being coil, turns or 310 helix. In this sense, the secondary structure topology is very similar to that of the `cysteinestabilized ab' (CSab) motif, typified by the scorpion toxin charybdotoxin and the defensins (Cornet et al., 1995), in which the a segment is a well defined a-helix. The disulfide pairings of the CSab fold are the same as those of the ICK motif, although charybdotoxin, for example, neither satisfies the motif consensus sequence nor contains a cystine knot. A series of antimicrobial and carbohydrate binding polypeptides with the reverse bbab topology and three or four disulfide bridges (AMP, hevein, chitinase and wheat germ agglutinin domains) have been compared to the ICK motif (Chagolla-Lopez et al., 1994). They have the ICK disulfide bridge pairings (for the first three disulfide bridges) and
Fig. 2. Schematic diagram of the ICK motif illustrating motif disul®de bridges as bold bars, additional disul®de bridges as shaded bars, half-cystine residues as C and b-strand residues that are not necessarily half-cystine residues as X.
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almost satisfy the consensus sequence (the second gap is longer by one residue), but do not contain the cystine knot (Wright, 1989; Martins et al., 1996). In this respect they serve as a useful reminder that sequence motifs are good indicators of likely structural similarity but are not absolutely prescriptive. A similar triple-stranded b-sheet to that of the ICK motif, but without the cystine knot, also occurs as a subset of the four-stranded, anti-parallel bsheet in the type I and II sea anemone toxins, typified by the anthopleurins and Sh I, respectively (Norton, 1991). This four-stranded b-sheet occurs in turn as a subset of the serine protease fold (Orengo et al., 1993). Figure 3 shows the structures of several representative examples of ICK polypeptides. o-CgTx GVIA is typical of the cone-shell and spider toxins, all of which have a doublet of half-cystine residues, and in particular of those members with a short loop between the fifth and sixth half-cystine residues. Conotoxin GS, a sodium channel inhibitor, has the shortest second loop (between the second and third half-cystine residues), only three residues in length. CPI, an example from the plant kingdom, has the largest gap (five residues) between the third and fourth half-cystine residues. The only known (nonredundant) example of a cyclic member, kalata B1, is also shown. The recently determined structure of the spider toxin o-ACTX-Hv1 has the longest fourth loop, of 13 residues. The structure of the Na+ channel modulator robustoxin, from the Sydney funnel-web spider, was solved recently in our laboratory (Pallaghy et al., 1997). The disulfide pairing
Fig. 3. MOLSCRIPT diagrams (Kraulis, 1991) of the structures of six typical ICK motif members discussed in the text (see caption to Fig. 1 for literature references). The motif disul®de bridges are shown in bold and the additional disul®de bridges in lighter shading.
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Fig. 4. Ribbon representation of the backbone of robustoxin with all side-chains shown, the half-cystine side-chains being shown as un®lled segments. The structure closest to the mean over the family of NMR-derived structures (Pallaghy et al., 1997) is shown. This diagram was created using Insight II (Molecular Simulations, San Diego).
pattern of robustoxin, which contains a triplet of half-cystine residues, was determined by a combination of NMR and chemical techniques. In Figs 3 and 4 it can be seen that the fourth disulfide bridge of robustoxin links the loop between the fourth and fifth halfcystine residues of the motif with a C-terminal extension, which consists of a series of gturns (or 310 helix in the highly homologous versutoxin (Fletcher et al., 1997a)). A similar length C-terminal extension exists in the case of o-Aga IVB (Fig. 3) but it is not stabilized by a disulfide bridge and is not recorded in the deposited coordinates (Yu et al., 1993). Instead, the fourth disulfide bridge of o-Aga IVB, mentioned previously, constrains the fifth loop, which is of exactly the same size as the corresponding O-loop of robustoxin. Many of the ICK motif structures, especially the ion-channel toxins, display highly basic surfaces, as exemplified by robustoxin (Fig. 4). In such cases it is suspected that these charged groups play a role in interactions between the toxin and the vestibule and pore of the target ion channel. Interestingly, in the case of robustoxin, the opposite face is dominated by hydrophobic residues (Fig. 4). The cellulose binding domains of cellobiohydrolases I and II (CBH-I and II) from the fungus Trichoderma reesei are interesting putative members of the ICK motif family (Fig. 5). The structure of the binding domain of CBH-I, which contains two disulfides, has been determined (Kraulis et al., 1989), and has the ICK fold, including the correct positions of the half-cystine residues in the triple-stranded b-sheet, except that the first disulfide bridge of the motif is absent. The structure of the highly homologous binding domain of CBH-II, with three disulfide bridges, has not yet been determined although it would be expected to be very similar to that of CBH-I. We suspect that the first disulfide bridge of CBH-II is analogous to the first disulfide bridge of the motif. However, if this is the case, then the last half-cystine of CBH-II would pair with Cys19 (to give the 1±4, 2±5, 3±6 motif pairing pattern), whereas it is known that the last half-cystine of the binding domain of CBH-I pairs instead with Cys20 (Fig. 5). If the experimental structure of the
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Fig. 5. Sequence alignment of the binding domains of CBH-I (GUX1_TRIRE, Shoemaker et al., 1983) and CBH-II (GUX2_TRIRE, Teeri et al., 1987) both from the fungus Trichoderma reesei (SWISS PROT accession codes in parentheses, with citations). The subset of the motif found in the tertiary structure of the binding domains of CBH-I is shown with bold disul®de bridge indications and b-strand arrows. The proposed disul®de pairings for the binding domain of CBH-II (whose disul®de pairings are not known speci®cally) that would cause it to have the motif disul®de pairings are shown as dashed lines, as are the expected b-strands.
binding domain of CBH-II confirms this, then the second and fourth consensus gaps would need to be modified to X3±9 and X1±5, respectively. The binding domain of CBH-II would represent the first ICK family member that, although an independently folding domain, is part of a multi-domain structure. In addition, these domains would also represent additional fungal members of the ICK family. FOLDING AND ENGINEERING
The ICK motif is robust with respect to the gap sizes between the half-cystines and the sequence variation among the non-bridging residues. It does not follow, of course, that every combination of amino acid sequence changes will produce a folded structure (even native o-conotoxins only fold in vitro with 16% to 50% efficiency, as shown by PriceCarter et al. (1996)). Intuitively, one might suspect that folding is directed by independent formation of the reverse turns, thus bringing the appropriate half-cystine residues in close proximity. Simulated folding studies (Pallaghy, unpublished data) suggest that such a folding mechanism occurs, involving co-operativity between the long-range (in sequence) interactions of disulfide formation and short-range b-turn formation. Thus, substitutions of single residues important for folding can greatly perturb the thermodynamics and/or kinetics of folding, as observed experimentally in alanine scan studies of o-CgTx GVIA (Kim et al., 1995; Lew et al., 1997). The motif appears to be an excellent compact globular scaffold for the presentation of a variety of functional groups, and has been used in nature to generate a range of polypeptides with diverse biological targets. Even quite subtle changes in amino acid sequence can lead to changes in target specificity. For example, the N-type calcium channel blocker o-CmTx MVIIA (Olivera et al., 1987) has a far higher sequence similarity (over 80%) to the P/Q-type calcium channel blocker o-CmTx MVIIC than it does to another N-type calcium channel blocker o-CgTx GVIA, which is of similar potency. Chiche et al. (1993) have demonstrated that it is possible to graft functional residues from one sequence onto another and create a chimeric ICK protein that retains both activities. They describe a chimera consisting of the N-terminal 27 residues from EETI-II (a trypsin inhibitor from Ecballium elaterium, highly homologous to CMTI-I in Fig. 1) and
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the C-terminal five residues from the carboxypeptidase inhibitor CPI (Fig. 1). It is also possible to transplant functional residues from other proteins into toxin structures, as in the case of a metal binding site similar to that in carbonic anhydrase, which has been engineered into charybdotoxin (Vita et al., 1995). Phage-display technology has the ability to generate improved potencies as well as arbitrary functionalities that would not be readily achievable using conventional peptide design and engineering (Cunningham and Wells, 1997). In several cases, structure±function studies of naturally occurring ICK motif molecules have demonstrated that the most important residues often originate from only one or two loops. For example, the key residues in o-CgTx GVIA lie in the loops between half-cystine residues one and two and between half-cystine residues two and three (Kim et al., 1995; Lew et al., 1997). As mentioned above, the important residues in CPI are located in the C-terminal region, after the final motif half-cystine residue (Chiche et al., 1993). This suggests a scenario in which certain combinations of loops could be randomized and selected for by phage-display and screening techniques. Further modification could then proceed with a different loop selected for randomization, with the rest of the molecule retaining the most potent sequence from the previous iteration. In conclusion, we have attempted to summarize the key structural features of the inhibitor cystine knot motif and to draw attention to the diversity of polypeptides that adopt this structural motif. As more examples are found in nature, it is likely that small changes will be required in the consensus sequence currently used to characterize the motif, and some of these changes have been foreshadowed herein. Cone snails and spiders appear to be rich sources of polypeptides based on this structural motif. However, it is likely that protein engineering and phage display technologies will contribute further to the range of molecules having this structure, and that commercial and therapeutic applications of these polypeptides will become more common. Acknowledgements ÐWe thank Jamie Fletcher and Justine Hill for providing the coordinates of o-atracotoxinHv1 and conotoxin GS, respectively, prior to their release from the Protein Data Bank. REFERENCES Adams, M. E., Mintz, I. M., Reily, M. D., Thanabal, V. and Bean, B. P. (1993) Structure and properties of o-agatoxin IVB, a new agonist of P-type calcium channels. Mol. Pharmacol. 44, 681±688. Arai, K., Ishima, R., Morikawa, S., Miyasaka, A., Imoto, T., Yoshimura, S., Aimoto, S. and Akasaka, K. (1995) Three-dimensional structure of gurmarin, a sweet taste-suppressing polypeptide. J. Biomol. NMR 5, 297±305. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. and Tasumi, M. (1977) The Protein Data Bank: A computer-based archival ®le for macromolecular structures. J. Mol. Biol. 112, 535±542. Berg, J. M. (1988) Proposed structure for the zinc-binding domain from trancription factor IIIA and related proteins. Proc. Natl. Acad. Sci. U.S.A. 85, 99±102. Broekaert, W. F., MarieÈn, W., Terras, F. R. G., De Bolle, M. F. C., Proost, P., Van Damme, J., Dillen, L., Claeys, M., Rees, S. B., Vanderleyden, J., Cammue, B. P. A. (1992) Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine rich domain of chitin-binding proteins. Biochemistry 31, 4308±4314. Brenner, S. E., Chothia, C. and Hubbard, T. J. P. (1997) Population statistics of protein structures: Lessons from structural classi®cations. Curr. Opin. Struct. Biol. 7, 369±376. Cammue, B. P. A., De Bolle, M. F. C., Terras, F. R. G., Proost, P., Van Damme, J., Rees, S. B., Vanderleyden, J. and Broekaert, W. F. (1992) Isolation and characterisation of a novel class of plant antimicrobial peptides from Mirabilis jalapa L. seeds. J. Biol. Chem. 267, 2228±2233. Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., SaÂnchez, R. and Pongor, S. (1994) A novel a-amylase inhibitor from Amaranth (Amaranthus hypocondriacus) seeds. J. Biol. Chem. 269, 23675±23680. Chiche, L., Heitz, A., Padilla, A., Le-Nguyen, D. and Castro, B. (1993) Solution conformation of a synthetic bis-headed inhibitor of trypsin and carboxypeptidase A: New structural alignment between the squash inhibitors and the potato carboxypeptidase inhibitor. Prot. Engin. 7, 675±682.
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A., Pettersson, G., Knowles, J. and Gronenborn, A. M. (1989) Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometrydynamical simulated annealing. Biochemistry 28, 7241±7257. Le Nguyen, D., Heitz, A., Chiche, L., Castro, B., Boigegrain, R. A., Favel, A. and Coletti-Previero, M. A. (1990) Molecular recognition between serine proteases and new bioactive microproteins with a knotted structure. Biochimie 72, 431±435. Lew, M. J., Flinn, J. P., Pallaghy, P. K., Murphy, R., Whorlow, S. L., Wright, C. E., Norton, R. S. and Angus, J. A. (1997) Structure-function relationships of o-conotoxin GVIA. Synthesis, structure, calcium channel binding, and functional assay of alanine-substituted analogues. J. Biol. Chem. 272, 12014±12023. Liang, S. P., Zhang, D. Y., Pan, X., Chen, Q. and Zhou, P. A. (1993) Properties and amino acid sequence of huwentoxin-I, a neurotoxin puri®ed from the venom of the Chinese bird spider Selenocosmia huwena. Toxicon 31, 969±978. Martins, J. C., Maes, D., Loris, R., Pepermans, H. A., Wyns, L., Willem, R. and Verheyden, P. (1996) 1 H NMR study of the solution structure of Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus. J. Mol. Biol. 258, 322±333. McDonald, N. Q. and Hendrickson, W. A. (1993) A structural superfamily of growth factors containing a cystine-knot motif. Cell 73, 421±424. McIntosh, J. M., Hasson, A., Spira, M. E., Gray, W. R., Li, W., Marsh, M., Hillyard, D. R. and Olivera, B. M. (1995) A new family of conotoxins that blocks voltage-gated sodium channels. J. Biol. Chem. 270, 16796±16802. Narasimhan, L., Singh, J., Humblet, C., Guruprasad, K. and Blundell, T. (1994) Snail and spider toxins share a similar tertiary structure and `cystine motif'. Nat. Struct. Biol. 1, 850±852. Newcomb, R., Palma, A., Fox, J., Gaur, S., Lau, K., Chung, D., Cong, R., Bell, J. R., Horne, B., Nadasdi, L. and Ramachandran, J. (1995) SNX-325, a novel calcium channel antagonist from the spider Segestria ¯orentina. Biochemistry 34, 8341±8347. Nilges, M., Habazettl, J., BruÈnger, A. T. and Holak, T. A. (1991) Relaxation matrix re®nement of the solution structure of squash trypsin inhibitor. J. Mol Biol. 248, 106±124. Norton, R. S. (1991) Structure and structure±function relationships of sea anemone proteins that interact with the sodium channel. Toxicon 29, 1051±1094. Olivera, B. M., McIntosh, J. M., Cruz, L. J., Luque, F. A. and Gray, W. R. (1984) Puri®cation and sequence of a presynaptic peptide toxin from Conus geographus venom. Biochemistry 23, 5087±5090.
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Olivera, B. M., Cruz, L. J., De Santos, V., Lecheminant, G. W., Grin, D., Zeikus, R. D., McIntosh, J. M., Galyean, R., Varga, J., Gray, W. R. and Rivier, J. E. (1987) Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using o-conotoxin from Conus magus venom. Biochemistry 26, 2086±2090. Omecinsky, D. O., Holub, K. E., Adams, M. E. and Reily, M. D. (1996) Three-dimensional structure analysis of m-agatoxins: Further evidence for common motifs among neurotoxins with diverse ion channel speci®cities. Biochemistry 35, 2836±2844. Orengo, C. A., Flores, T. P., Jones, D. T., Taylor, W. R. and Thornton, J. M. (1993) Recurring structural motifs in proteins with dierent functions. Curr. Biol. 3, 131±139. Pallaghy, P. K., Nielsen, K. J., Craik, D. J. and Norton, R. S. (1994) A common structural motif incorporating a cystine knot and triple-stranded b-sheet in toxic and inhibitory polypeptides. Protein Sci. 3, 1833± 1839. Pallaghy, P. K., Alewood, D., Alewood, P. F. and Norton, R. S. (1997) Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus. FEBS Lett. 419, 191±196. Price-Carter, M., Gray, W. R. and Goldenberg, D. P. (1996) Folding of o-conotoxins. 1. Ecient disul®decoupled folding of mature sequences in vitro. Biochemistry 35, 15537±15546. Qu, Y., Liang, S., Ding, J., Liu, X., Zhang, R. and Gu, X. (1997) Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spider Selenocosmia huwena: 2. Three-dimensional structure in solution. J. Protein Chem. 16, 565±574. Rees, D. C. and Lipscomb, W. N. (1982) Re®ned crystal structure of the potato inhibitor complex of carboxypeptidase A at 2.5 AÊ. J. Mol. Biol. 160, 475±498. Saether, O., Craik, D. J., Campbell, I. D., Sletten, K., Juul, J. and Norman, D. G. (1995) Elucidation of the primary and three-dimensional structure of the uterotonic polypeptide kalata B1. Biochemistry 34, 4147± 4158. Scanlon, M. J., Naranjo, D., Thomas, L., Alewood, P. F., Lewis, R. J. and Craik, D. J. (1997) Solution structure and proposed binding mechanism of a novel potassium channel toxin k-conotoxin PVIIA. Structure 5, 1585±1597. Sheumack, D. D., Claassens, R., Whiteley, N. M. and Howden, M. E. H. (1985) Complete amino acid sequence of a new type of lethal neurotoxin from the venom of the funnel-web spider Atrax robustus. FEBS Lett. 181, 154±156. Shoemaker, S., Schweickart, V., Ladner, M., Glefand, D., Kwok, S., Myambo, K. and Innis, M. (1983) Molecular cloning of exo-cellobiohydrolase I derived from Trichoderma reesei strain L27. Biotechnology 1, 691±696. Shon, K.-J., Hasson, A., Spira, M. E., Cruz, L. J., Gray, W. R. and Olivera, B. M. (1994) d-conotoxin GmVIA, a novel peptide from the venom of Conus gloriamaris. Biochemistry 33, 11420±11425. Skinner, W. S., Adams, M. E., Quistad, G. B., Kataoka, H., Cesarin, B. J., Enderlin, F. E. and Schooley, D. A. (1989) Puri®cation and characterisation of two classes of neurotoxins from the funnel web spider, Agelenopsis aperta. J. Biol. Chem. 264, 2150±2155. Teeri, T. T., Lehtovaara, P., Kauppinen, S., Salovuori, I. and Knowles, J. (1987) Homologous domains in Trichoderma reesei cellulolytic enzymes: Gene sequence and expression of cellobiohydrolase II. Gene 51, 43± 52. Terlau, H., Shon, K.-J., Grilley, M., Stocker, M., StuÈhmer, W. and Olivera, B. M. (1996) Strategy for rapid immobilisation of prey by a ®sh-hunting marine snail. Nature 381, 148±151. Van Kan, J. A. L., Van Den Ackerveken, G. F. J. M. and De Wit, P. J. G. M. (1991) Cloning and characterization of avirulence gene avr9 of the fungal pathogen Cladosporium fulvum, causal agent of tomato leaf mold. Mol. Plant Microb. Interact. 4, 52±59. Vervoort, J., Van Den Hooven, H. W., Berg, A., Vossen, P., Vogelsang, R., Joosten, M. H. A. J. and De Wit, P. J. G. M. (1997) The race-speci®c elicitor AVR9 from the tomato pathogen Cladosporium fulvum: A cystine knot protein. Sequence-speci®c 1 H NMR assignments, secondary structure and global fold of the protein. FEBS Lett. 404, 153±158. Vita, C., Roumestand, C., Toma, F. and Menez, A. (1995) Scorpion toxins as natural scaolds for protein engineering. Proc. Natl. Acad. Sci. U.S.A. 92, 6404±6408. Wilusz, T., Wieczorek, M., Polanowski, A., Denton, A., Cook, J. and Laskowski, M., Jr. (1983) Amino-acid sequence of two trypsin inhibitors ITD I and ITD III from squash seeds (Cucurbita maxima). Hoppe± Seyler's Z. Physiol. Chem. 364, 93±95. Wright, C. S. (1989) Comparison of the re®ned crystal structures of two wheat germ isolectins. J. Mol. Biol. 209, 475±487. Yanagawa, Y., Abe, T., Satake, M., Odani, S., Suzuki, J. and Ishikawa, K. (1988) A novel sodium channel inhibitor from Conus geographus: Puri®cation, structure, and pharmacological properties. Biochemistry 27, 6256±6262. Yu, H., Rosen, M. K., Saccomano, N. A., Phillips, D., Volkmann, R. A. and Schreiber, S. L. (1993) Sequential assignment and structure determination of spider toxin o-Aga-IVB. Biochemistry 32, 13123± 13129.
Toxicon Vol. 36, No. 11, pp. 1573±1583, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0041-0101/98 $19.00 + 0.00
THE CYSTINE KNOT STRUCTURE OF ION CHANNEL TOXINS AND RELATED POLYPEPTIDES RAYMOND S. NORTON* and PAUL K. PALLAGHY Biomolecular Research Institute, 343 Royal Parade, Parkville, 3052, Australia (Received 12 February 1998; accepted 10 April 1998)
R. S. Norton and P. K. Pallaghy. The cystine knot structure of ion channel toxins and related polypeptides. Toxicon 36, 1573±1583, 1998.ÐAn increasing number of ion channel toxins and related polypeptides have been found to adopt a common structural motif designated the inhibitor cystine knot motif (Pallaghy P. K., Nielsen, K. J., Craik, D. J., Norton, R. S. (1994) A common structural motif incorporating a cystine knot and triple-stranded bsheet in toxic and inhibitory polypeptides. Protein Science 3, 1833±1839). These globular, disul®de-stabilized molecules come from phylogenetically diverse sources, including spiders, cone shells, plants and fungi, and have various functions, although many target voltage-gated ion-channels. The common motif consists of a cystine knot and a triple-stranded, anti-parallel b-sheet. Examples of ion-channel toxins known to adopt this structure are the o-conotoxins and o-agatoxins, and, more recently, robustoxin, versutoxin and protein 5 from spiders, as well as k-conotoxin PVIIA and conotoxin GS from cone shells. The variations on the motif structure exempli®ed by these structures are described here. We also consider the sequences of several polypeptides that might adopt this fold, including SNX-325 from a spider, d-conotoxin PVIA and the mO-conotoxins from cone shells, and various plant and fungal polypeptides. The interesting case of the two- and three-disul®de bridged binding domains of the cellobiohydrolases from the fungus Trichoderma reesei is also discussed. The compact and robust nature of this motif makes it an excellent scaold for the design and engineering of novel polypeptides with enhanced activity against existing targets, or with activity against novel targets. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
It has been suggested that the number of protein families (that is, groups of proteins with recognizable sequence similarity) to be found in nature is about 1000, and that the number of unique protein folds is even lower (Brenner et al., 1997). Among small, disulfide crosslinked polypeptides, several different folds have been recognized. One that is found in an increasing number of small toxic and inhibitory polypeptides with diverse functions and origins is the inhibitor cystine knot (ICK) structural motif (Narasimhan et al., 1994; * Author to whom correspondence should be addressed. Email: [email protected] 1573
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Pallaghy et al., 1994). Polypeptides that adopt this structure have a range of interesting biological activities, several of which are relevant to the development of new human therapeutics. They are also of interest as scaffolds for protein engineering and phage display. In this article, we describe several recently-determined structures that share this motif, compare the motif with structural motifs found in other disulfide cross-linked polypeptides, and discuss additional polypeptide sequences that would be expected to adopt this motif structure. The ICK structural motif is characterised by a triple-stranded, anti-parallel b-sheet stabilized by a cystine knot. In terms of amino acid sequence, all known examples of this fold satisfy the following consensus: CX3±7CX3±6CX0±5CX1±4CX4±13C, where X can be any amino acid. The ICK motif is distinct from the growth factor cystine knot motif, found in transforming growth factor-b2, platelet-derived growth factor and nerve growth factor (McDonald and Hendrickson, 1993; Isaacs, 1995). It includes several plant protease inhibitors described as knottins (Le Nguyen et al., 1990), but expands the definition beyond these proteins and provides a more representative description of the motif. Structural motifs are known to occur in molecules with different functions and from diverse sources (Orengo et al., 1993). However, in the case of the ICK motif, the similarity goes beyond a simple topological similarity as it also involves the half-cystine residues occurring in fixed positions relative to the b-sheet hydrogen bonding pattern. This is reminiscent of sequence motifs such as the zinc finger motif (Berg, 1988), except that these motifs are predictive for a specific function and the sequence examples retain some homology among non-cystine residues. In the ICK motif this is not the case, and the functions are quite diverse. The fold, presumably dictated largely by the gaps between the half-cystine residues and their pairing pattern, appears to be an ideal compact globular scaffold for the presentation of a variety of functional groups, thereby generating a range of polypeptides with diverse biological targets. An interesting protein engineering application has already been demonstrated in the case of a chimera of a trypsin inhibitor and a carboxypeptidase inhibitor that retains both functions (see below). Exploitation of the ICK motif by phage display technology promises to create new biological activities with high potency. SEQUENCES
Figure 1 presents a non-redundant sequence alignment of representative members of the inhibitor cystine knot motif with distinct molecular targets and experimentally determined tertiary structures. These sequences come from organisms as diverse as spiders, coneshells, plants and fungi. As indicated above, they all satisfy the consensus sequence CX3ÿ7 CX3ÿ6 CX0ÿ5 CX1ÿ4 CX4ÿ13 C where X can be any amino acid (including half-cystine). An earlier version of this description, using the consensus sequence CX3±7CX4±6CX0±5CX1±4CX4±10C, enabled a search of amino acid sequence data bases to identify three polypeptides from diverse sources that should adopt this fold (Pallaghy et al., 1994). These were gurmarin, a sweet taste suppressor from the tree Gymnema sylvestre, huwentoxin-I, from the Chinese birdeating spider Selenocosmia huwena, and the race-specific elicitor AVR9, from the fungal tomato pathogen Cladosporium fulvum. Solution structures have since been reported for all three of these (Arai et al., 1995; Qu et al., 1997; Vervoort et al., 1997), and they confirm the predicted fold. A number of hits were also found in larger proteins but these were not
Fig. 1. Sequence alignment of members of the ICK motif for which experimentally determined structures are available and which have non-redundant sequences or distinct functions. The half-cystine residues of the motif are highlighted in bold. The continuous lines represent the motif disul®de bridges and the dashed lines the additional bridges. The arrows represent the typical motif b-strands. The SWISS-PROT or PIR database accession codes (the latter indicated by `PIR') and literature references are given for the amino acid sequences of all polypeptides shown here, followed by the Brookhaven Protein Data Bank (Bernstein et al., 1977) accession codes (where available), and literature references for the structures, as follows: robustoxin from Atrax robustus (TXRO_ATRRO, Sheumack et al., 1985; 1qdp, Pallaghy et al., 1997), o-ACTX-Hv1 from Hadronyche versuta (TXO6_HADVE; 1axh, Fletcher et al., 1997b), o-Aga IVB from Agelenopsis aperta (TX4B_AGEAP, Adams et al., 1993; 1oma, Yu et al., 1993), m-Aga I from Agelenopsis aperta (TXM1_AGEAP, Skinner et al., 1989; 1eit, Omecinsky et al., 1996), huwentoxin-I from Selenocosmia huwena (PIR-A37479, Liang et al., 1993; Qu et al., 1997), protein 5 from Brachypelma smithii (TXP5_BRASM, Kaiser et al., 1994; Hill et al., unpublished), o-CgTx GVIA from Conus geographus (CXO6_CONGE, Olivera et al., 1984; 2CCO, Pallaghy, and Norton, submitted), o-CmTx MVIIC from Conus magus (CXOC_CONMA, Hillyard et al., 1992; 1omn, Farr-Jones et al., 1995), k-CpTx PVIIA from Conus purpurascens (Terlau et al., 1996; 1av3, Scanlon et al., 1997), conotoxin GS from Conus geographus (CXGS_CONGE, Yanagawa et al., 1988; 1ag7, Hill et al., 1997), CMTI-I from Cucurbita maxima (ITR1_CUCMA, Wilusz et al., 1983; 3cti, Nilges et al., 1991), CPI from Solanum tuberosum (MCPI_SOLTU, Hass et al., 1975; 4cpa, Rees and Lipscomb, 1982), gurmarin from Gymnema sylvestre (PIR-JX0200, Kamei et al., 1992; 1gur, Arai et al., 1995), kalata B1 from Oldenlandia anis (PIR-A56283, Saether et al., 1995; 1kal, Saether et al., 1995), and AVR9 from Cladosporium fulvum (AVR9_CLAFU, Van Kan et al., 1991; Vervoort et al., 1997).
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considered further, usually because these proteins contained several additional halfcystines and it was not clear that the half-cystines which satisfied the motif consensus were linked to one another. However, as discussed below for cellobiohydrolase, it is certainly possible for small domains of large proteins to adopt the motif structure. The third and fourth half-cystines of the motif are usually contiguous, and always so for spider and cone-shell polypeptides. Gurmarin, however, is the only currently known representative from the plant kingdom that satisfies this criterion. Interestingly, the fungal polypeptide AVR9, which induces defence responses in plants resistant to the producing fungus, resembles the plant protease inhibitors and kalata in having a finite number of residues between the third and fourth half-cystines, although the functional significance of this is unknown at present. Most of the members have only the three disulfide bridges that comprise the cystine knot, although there are three (nonredundant) examples that contain a fourth disulfide bridge: robustoxin (otherwise known as d-ACTX-Hr1), o-agatoxin IVB (o-Aga-IVB) and m-agatoxin I (m-Aga-I). Robustoxin has an unusual disulfide pairing pattern, characterised in the sequence by a triplet of half-cystine residues, the first two being the third and fourth of the cystine knot motif. The additional disulfide bridge links the loop between the fourth and fifth half-cystine residues to a C-terminal extension of the sequence. o-Aga-IVB and m-Aga-I have an additional disulfide bridge that stabilizes the fifth loop and is located between the fifth and sixth half-cystine residues of the motif. Several amino acid sequences have been reported recently that either satisfy or closely resemble the consensus sequence. d-conotoxin PVIA, from the fish-hunting cone shell Conus purpurascens, has novel `lockjaw' activity in fish due to its retardation of sodium channel inactivation (Shon et al., 1994; Terlau et al., 1996) and satisfies the consensus sequence. The mO-conotoxins MrVIA and MrVIB, from the cone shell Conus marmoreus, are sodium channel and L-type calcium channel inhibitors (Fainzilber et al., 1995; McIntosh et al., 1995). If they adopt the motif structure, this would require an expansion of the second gap size, from X3±6 to X3±9. SNX-325, from the spider Segestria florentina, probably inhibits N-type calcium channels (Newcomb et al., 1995) and would require a major revision of the last gap size (from X4±13 to X4±20). If confirmed as a new member of the motif, it would contain a larger, but otherwise similar, disulfide-stabilized fifth loop, as found in o-Aga-IVB and m-Aga-I. The existing member of the family with the largest last gap (13 residues) is o-ACTX-Hv1 which, however, has no additional disulfide bridge. The insect a-amylase inhibitor from the Mexican crop plant Amaranthus hypocondriacus satisfies the consensus sequence and was in fact homology modelled using a trypsin inhibitor from Ecballium elaterium (highly homologous to CMT-I in Fig. 1) and the binding domain of cellobiohydrolase I (see below) (Chagolla-Lopez et al., 1994). The chitin binding polypeptides, AMP1 and AMP2, from the plant Mirabilis jalapa, inhibit the growth of plant pathogenic fungi (Broekaert et al., 1992; Cammue et al., 1992). If they were shown to be members of the ICK motif structural family, the second gap size would have to be expanded from X3±6 to X3±8, and the motif would then be endowed with members from plants that attack fungi and vice-versa. The naming of the motif (Pallaghy et al., 1994) continues to be appropriate in terms of the known functions of the majority of polypeptides in this structural family. Both the spider and cone-shell sequences include blockers for the three major voltage-gated ion channel types (Na+, K+ and Ca2+). Neither of the plant kingdom members with known molecular targets is an ion-channel blocker; rather, both are protease inhibitors. The putative plant and fungal members of the motif include other enzyme inhibitors and binding polypeptides. Some new members of the family, for example robustoxin, modulate
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rather than block the function of their target receptor, in this case the voltage-gated Na+ channel, but as a general descriptor the name is still adequate. STRUCTURES
The motif structure (Fig. 2) consists of an anti-parallel, triple-stranded b-sheet stabilized by a cystine knot (Narasimhan et al., 1994; Pallaghy et al., 1994). The cystine knot is comprised of a ring formed by two disulfide bridges and the interconnecting backbone, through which the third disulfide passes. The first b-strand of the motif is typically three residues in length with the third position being a half-cystine residue, although the strand can be highly distorted or linked to the sheet by only a single b-bridge, as in robustoxin, oACTX-Hv1 and conotoxin GS. The later two b-strands form a hairpin structure, with the strand lengths typically being three and four residues, respectively. Half-cystine residues fill positions two and three, respectively, of b-strands II and III. The defining characteristics of the motif up to this point (Pallaghy et al., 1994) are that these later two half-cystine residues pack against each other in the b-sheet, and the half-cystine of bstrand III (being the central strand) packs against the first residue of b-strand I (two residues preceding the half-cystine of b-strand I). The ICK motif topology is babb-like, with the a segment being coil, turns or 310 helix. In this sense, the secondary structure topology is very similar to that of the `cysteinestabilized ab' (CSab) motif, typified by the scorpion toxin charybdotoxin and the defensins (Cornet et al., 1995), in which the a segment is a well defined a-helix. The disulfide pairings of the CSab fold are the same as those of the ICK motif, although charybdotoxin, for example, neither satisfies the motif consensus sequence nor contains a cystine knot. A series of antimicrobial and carbohydrate binding polypeptides with the reverse bbab topology and three or four disulfide bridges (AMP, hevein, chitinase and wheat germ agglutinin domains) have been compared to the ICK motif (Chagolla-Lopez et al., 1994). They have the ICK disulfide bridge pairings (for the first three disulfide bridges) and
Fig. 2. Schematic diagram of the ICK motif illustrating motif disul®de bridges as bold bars, additional disul®de bridges as shaded bars, half-cystine residues as C and b-strand residues that are not necessarily half-cystine residues as X.
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almost satisfy the consensus sequence (the second gap is longer by one residue), but do not contain the cystine knot (Wright, 1989; Martins et al., 1996). In this respect they serve as a useful reminder that sequence motifs are good indicators of likely structural similarity but are not absolutely prescriptive. A similar triple-stranded b-sheet to that of the ICK motif, but without the cystine knot, also occurs as a subset of the four-stranded, anti-parallel bsheet in the type I and II sea anemone toxins, typified by the anthopleurins and Sh I, respectively (Norton, 1991). This four-stranded b-sheet occurs in turn as a subset of the serine protease fold (Orengo et al., 1993). Figure 3 shows the structures of several representative examples of ICK polypeptides. o-CgTx GVIA is typical of the cone-shell and spider toxins, all of which have a doublet of half-cystine residues, and in particular of those members with a short loop between the fifth and sixth half-cystine residues. Conotoxin GS, a sodium channel inhibitor, has the shortest second loop (between the second and third half-cystine residues), only three residues in length. CPI, an example from the plant kingdom, has the largest gap (five residues) between the third and fourth half-cystine residues. The only known (nonredundant) example of a cyclic member, kalata B1, is also shown. The recently determined structure of the spider toxin o-ACTX-Hv1 has the longest fourth loop, of 13 residues. The structure of the Na+ channel modulator robustoxin, from the Sydney funnel-web spider, was solved recently in our laboratory (Pallaghy et al., 1997). The disulfide pairing
Fig. 3. MOLSCRIPT diagrams (Kraulis, 1991) of the structures of six typical ICK motif members discussed in the text (see caption to Fig. 1 for literature references). The motif disul®de bridges are shown in bold and the additional disul®de bridges in lighter shading.
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Fig. 4. Ribbon representation of the backbone of robustoxin with all side-chains shown, the half-cystine side-chains being shown as un®lled segments. The structure closest to the mean over the family of NMR-derived structures (Pallaghy et al., 1997) is shown. This diagram was created using Insight II (Molecular Simulations, San Diego).
pattern of robustoxin, which contains a triplet of half-cystine residues, was determined by a combination of NMR and chemical techniques. In Figs 3 and 4 it can be seen that the fourth disulfide bridge of robustoxin links the loop between the fourth and fifth halfcystine residues of the motif with a C-terminal extension, which consists of a series of gturns (or 310 helix in the highly homologous versutoxin (Fletcher et al., 1997a)). A similar length C-terminal extension exists in the case of o-Aga IVB (Fig. 3) but it is not stabilized by a disulfide bridge and is not recorded in the deposited coordinates (Yu et al., 1993). Instead, the fourth disulfide bridge of o-Aga IVB, mentioned previously, constrains the fifth loop, which is of exactly the same size as the corresponding O-loop of robustoxin. Many of the ICK motif structures, especially the ion-channel toxins, display highly basic surfaces, as exemplified by robustoxin (Fig. 4). In such cases it is suspected that these charged groups play a role in interactions between the toxin and the vestibule and pore of the target ion channel. Interestingly, in the case of robustoxin, the opposite face is dominated by hydrophobic residues (Fig. 4). The cellulose binding domains of cellobiohydrolases I and II (CBH-I and II) from the fungus Trichoderma reesei are interesting putative members of the ICK motif family (Fig. 5). The structure of the binding domain of CBH-I, which contains two disulfides, has been determined (Kraulis et al., 1989), and has the ICK fold, including the correct positions of the half-cystine residues in the triple-stranded b-sheet, except that the first disulfide bridge of the motif is absent. The structure of the highly homologous binding domain of CBH-II, with three disulfide bridges, has not yet been determined although it would be expected to be very similar to that of CBH-I. We suspect that the first disulfide bridge of CBH-II is analogous to the first disulfide bridge of the motif. However, if this is the case, then the last half-cystine of CBH-II would pair with Cys19 (to give the 1±4, 2±5, 3±6 motif pairing pattern), whereas it is known that the last half-cystine of the binding domain of CBH-I pairs instead with Cys20 (Fig. 5). If the experimental structure of the
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Fig. 5. Sequence alignment of the binding domains of CBH-I (GUX1_TRIRE, Shoemaker et al., 1983) and CBH-II (GUX2_TRIRE, Teeri et al., 1987) both from the fungus Trichoderma reesei (SWISS PROT accession codes in parentheses, with citations). The subset of the motif found in the tertiary structure of the binding domains of CBH-I is shown with bold disul®de bridge indications and b-strand arrows. The proposed disul®de pairings for the binding domain of CBH-II (whose disul®de pairings are not known speci®cally) that would cause it to have the motif disul®de pairings are shown as dashed lines, as are the expected b-strands.
binding domain of CBH-II confirms this, then the second and fourth consensus gaps would need to be modified to X3±9 and X1±5, respectively. The binding domain of CBH-II would represent the first ICK family member that, although an independently folding domain, is part of a multi-domain structure. In addition, these domains would also represent additional fungal members of the ICK family. FOLDING AND ENGINEERING
The ICK motif is robust with respect to the gap sizes between the half-cystines and the sequence variation among the non-bridging residues. It does not follow, of course, that every combination of amino acid sequence changes will produce a folded structure (even native o-conotoxins only fold in vitro with 16% to 50% efficiency, as shown by PriceCarter et al. (1996)). Intuitively, one might suspect that folding is directed by independent formation of the reverse turns, thus bringing the appropriate half-cystine residues in close proximity. Simulated folding studies (Pallaghy, unpublished data) suggest that such a folding mechanism occurs, involving co-operativity between the long-range (in sequence) interactions of disulfide formation and short-range b-turn formation. Thus, substitutions of single residues important for folding can greatly perturb the thermodynamics and/or kinetics of folding, as observed experimentally in alanine scan studies of o-CgTx GVIA (Kim et al., 1995; Lew et al., 1997). The motif appears to be an excellent compact globular scaffold for the presentation of a variety of functional groups, and has been used in nature to generate a range of polypeptides with diverse biological targets. Even quite subtle changes in amino acid sequence can lead to changes in target specificity. For example, the N-type calcium channel blocker o-CmTx MVIIA (Olivera et al., 1987) has a far higher sequence similarity (over 80%) to the P/Q-type calcium channel blocker o-CmTx MVIIC than it does to another N-type calcium channel blocker o-CgTx GVIA, which is of similar potency. Chiche et al. (1993) have demonstrated that it is possible to graft functional residues from one sequence onto another and create a chimeric ICK protein that retains both activities. They describe a chimera consisting of the N-terminal 27 residues from EETI-II (a trypsin inhibitor from Ecballium elaterium, highly homologous to CMTI-I in Fig. 1) and
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the C-terminal five residues from the carboxypeptidase inhibitor CPI (Fig. 1). It is also possible to transplant functional residues from other proteins into toxin structures, as in the case of a metal binding site similar to that in carbonic anhydrase, which has been engineered into charybdotoxin (Vita et al., 1995). Phage-display technology has the ability to generate improved potencies as well as arbitrary functionalities that would not be readily achievable using conventional peptide design and engineering (Cunningham and Wells, 1997). In several cases, structure±function studies of naturally occurring ICK motif molecules have demonstrated that the most important residues often originate from only one or two loops. For example, the key residues in o-CgTx GVIA lie in the loops between half-cystine residues one and two and between half-cystine residues two and three (Kim et al., 1995; Lew et al., 1997). As mentioned above, the important residues in CPI are located in the C-terminal region, after the final motif half-cystine residue (Chiche et al., 1993). This suggests a scenario in which certain combinations of loops could be randomized and selected for by phage-display and screening techniques. Further modification could then proceed with a different loop selected for randomization, with the rest of the molecule retaining the most potent sequence from the previous iteration. In conclusion, we have attempted to summarize the key structural features of the inhibitor cystine knot motif and to draw attention to the diversity of polypeptides that adopt this structural motif. As more examples are found in nature, it is likely that small changes will be required in the consensus sequence currently used to characterize the motif, and some of these changes have been foreshadowed herein. Cone snails and spiders appear to be rich sources of polypeptides based on this structural motif. However, it is likely that protein engineering and phage display technologies will contribute further to the range of molecules having this structure, and that commercial and therapeutic applications of these polypeptides will become more common. Acknowledgements ÐWe thank Jamie Fletcher and Justine Hill for providing the coordinates of o-atracotoxinHv1 and conotoxin GS, respectively, prior to their release from the Protein Data Bank. REFERENCES Adams, M. E., Mintz, I. M., Reily, M. D., Thanabal, V. and Bean, B. P. (1993) Structure and properties of o-agatoxin IVB, a new agonist of P-type calcium channels. Mol. Pharmacol. 44, 681±688. Arai, K., Ishima, R., Morikawa, S., Miyasaka, A., Imoto, T., Yoshimura, S., Aimoto, S. and Akasaka, K. (1995) Three-dimensional structure of gurmarin, a sweet taste-suppressing polypeptide. J. Biomol. NMR 5, 297±305. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. and Tasumi, M. (1977) The Protein Data Bank: A computer-based archival ®le for macromolecular structures. J. Mol. Biol. 112, 535±542. Berg, J. M. (1988) Proposed structure for the zinc-binding domain from trancription factor IIIA and related proteins. Proc. Natl. Acad. Sci. U.S.A. 85, 99±102. Broekaert, W. F., MarieÈn, W., Terras, F. R. G., De Bolle, M. F. C., Proost, P., Van Damme, J., Dillen, L., Claeys, M., Rees, S. B., Vanderleyden, J., Cammue, B. P. A. (1992) Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine rich domain of chitin-binding proteins. Biochemistry 31, 4308±4314. Brenner, S. E., Chothia, C. and Hubbard, T. J. P. (1997) Population statistics of protein structures: Lessons from structural classi®cations. Curr. Opin. Struct. Biol. 7, 369±376. Cammue, B. P. A., De Bolle, M. F. C., Terras, F. R. G., Proost, P., Van Damme, J., Rees, S. B., Vanderleyden, J. and Broekaert, W. F. (1992) Isolation and characterisation of a novel class of plant antimicrobial peptides from Mirabilis jalapa L. seeds. J. Biol. Chem. 267, 2228±2233. Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., SaÂnchez, R. and Pongor, S. (1994) A novel a-amylase inhibitor from Amaranth (Amaranthus hypocondriacus) seeds. J. Biol. Chem. 269, 23675±23680. Chiche, L., Heitz, A., Padilla, A., Le-Nguyen, D. and Castro, B. (1993) Solution conformation of a synthetic bis-headed inhibitor of trypsin and carboxypeptidase A: New structural alignment between the squash inhibitors and the potato carboxypeptidase inhibitor. Prot. Engin. 7, 675±682.
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