Critical Review. Structural Studies of Conotoxins

Transcription

1 IUBMB Life, 61(2): , February 2009 Critical Review Structural Studies of Conotoxins Norelle L. Daly and David J. Craik The University of Queensland, Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, Brisbane, QLD, Australia Summary Conotoxins are small disulfide-rich peptides from the venoms of marine cone snails. They target a variety of ion channels, transporters, and receptors besides the interest in their natural functions in venoms and they are of much interest as drug leads. This short article gives an overview of the structural diversity of conotoxins, and illustrates this diversity with recent selected examples of conotoxin structures. Ó 2009 IUBMB IUBMB Life, 61(2): , 2009 Keywords Conus; cystine knot; disulfide isomers; NMR; toxins. INTRODUCTION Conopeptides, From the Ocean to the Clinic Conopeptides are small peptides isolated from the venom of marine molluscs of the genus Conus and have long held the interest of biologists, pharmacologists, and structural biologists (1). This interest stems from the exquisite potency and selectivity that conotoxins display for a range of ion channels and receptors, making them excellent pharmacological probes as well as lead molecules in drug design (2). One conopeptide, x- MVIIA is currently marketed as Prialt 1 (generic name ziconotide) for the treatment of chronic pain (3). Several others are in various stages of clinical trials, including an analog of v-mria (4) and contulakin-g (5), for the treatment of chronic and neuropathic pain. At least three other conopeptides are currently undergoing preclinical evaluation for pharmaceutical applications, suggesting that the pipeline of potential conopeptide therapeutics is looking promising. Received 13 August 2008; accepted 1 November 2008 Address correspondence to: Dr. Norelle L. Daly, The University of Queensland, Institute for Molecular Bioscience, Carmody Road, Brisbane, Queensland 4072, Australia. Tel: Fax: n.daly@imb.uq.edu.au With up to 700 species of cone snails thought to occur in the world s oceans, and up to 200 peptides per venom, it has been estimated that more than 100,000 different conopeptides may exist (6 8). To date, several hundred conopeptides have been characterized and a systematic naming scheme has been developed (9). Conopeptides are broadly grouped into two classes, namely those with or without multiple disulfide bonds (6); the term conotoxin is used to describe the Conus peptides that are disulfide-rich, and the others are more generally referred to as conopeptides. Conopeptides have been categorized into families based primarily on their pharmacology and these families have been grouped into superfamilies. Individual families contain peptides that have a particular disulfide framework and biological target. The naming convention for individual peptides involves a Greek letter to indicate the pharmacological activity, one or two letters to indicate the source species from which the peptide was first isolated (eg M for Conus magus, Mr for Conus marmoreus to remove ambiguity), a Roman numeral to indicate the disulfide framework category and an upper case letter to denote the order of discovery within that category. For instance, x-conotoxin MVIIA, belongs to the x- conotoxin family, was isolated from Conus magus and was the first peptide discovered with framework VII. When novel peptides are discovered and the molecular target is not known a different convention is used, which consists of the species code in lower case, an Arabic numeral to designate the Cys pattern and a lower case letter to indicate a particular variant (1). A recent example is r11a, so named when first isolated from the fishhunting species Conus radiatus (10) but now designated as i- RXIA based on the discovery that it targets the Na V 1.6 subtype of the voltage-gated sodium channel (11). Scope of Review Conotoxins bind their target receptors with unparalleled specificity as a result of their well defined structures and high structural diversity. This structural diversity is the focus of this short review. Although there is a vast array of conotoxin structures, this review selects examples that highlight some of the ISSN print/issn online DOI: /iub.158

2 STRUCTURAL STUDIES OF CONOTOXINS 145 common motifs as well as some of the interesting features of conotoxin structures involving the influence of disulfide bonds on structural folds and dynamics. A discussion of the methods used in the structural analysis of conotoxins is also given. There have been a number of other recent reviews on conotoxins, including those that have focussed on their therapeutic potential (2, 12, 13), their structural features (14, 15), their chemical modifications (16), their specific pharmacological features (17), or on particular classes of conotoxins such as the a-conotoxins (18 20). In addition, there have been several earlier comprehensive reviews by the founder of the conotoxin field, Professor Baldomero Olivera (9, 21). The reader is referred to these various reviews for a more detailed background. Conotoxin Sequence Diversity To appreciate the structural diversity of conotoxins, it is necessary to appreciate their vast sequence diversity. Although conotoxins are generally restricted to~10 40 residues and the majority contains two or three disulfide bonds, there is still considerable scope for sequence variation, disulfide framework variation, and additional complexity introduced by the large number of post-translational modifications that are characteristic of the conopeptide family (1, 9, 22). The use of MS/MS techniques has recently aided the discovery of these novel sequences and post-translational modifications (23, 24). A selection of conopeptides is shown in Fig. 1 to illustrate these features. Contulakin G (5) is an example that does not contain disulfide bonds. Two examples of conotoxins displaying two disulfide bonds are shown, namely a-conotoxin PnIA (25) and v-conotoxin MrIA (4), which despite having a similar number of residues and four cysteine residues, have a different disulfide connectivity. For two-disulfide bonded conotoxins there are three possible connectivities, termed the globular, ribbon, and beads forms (26). The a- and v-conotoxins exhibit globular and ribbon disulfide connectivities respectively, but to date no native examples of beads conotoxins have been reported. Given the enormous number of predicted peptides in Conus venom it may be just a matter of time before an example of a peptide with a beads connectivity is discovered. Examples of conotoxins with more than two disulfide bonds include the abovementioned conotoxin MVIIA (27), and RXIA (10, 11), which contain three and four disulfide bonds, respectively (Fig. 1). There are numerous examples of three disulfide containing conotoxins that are distinguished not only by their sequence diversity but also by variations in the cysteine spacing and disulfide connectivity. By contrast, only a limited number of conotoxins with four or five disulfide bonds is currently known. However, the complexity of the conotoxin family has recently been expanded with the discovery of ~11 kda conotoxins that exist as dimers and have 10 cysteine residues per monomer (28). An additional phenomenon that expands the sequence diversity of conotoxins is the large number of post-translational modifications that have been observed (29). These modifications include D-amino acids, hydroxyproline, and c-carboxyglutamic acid residues amongst a host of others, with novel modifications being discovered on a regular basis. Despite the prevalence of these post-translational modifications, in general their function remains unknown. Conotoxin Targets and Therapeutic Applications A consequence of the sequence diversity of conotoxins is that they have a wide range of targets, including calcium channels, sodium channels, nicotinic acetylcholine receptors, the noradrenaline transporter, the NMDA receptor, and the neurotensin receptor (30). Despite this diverse range of targets, which appear to be present to maximize the efficiency of the venom, the therapeutic potential of a range of conotoxins is currently centered on the treatment of pain. For instance, N-type voltage sensitive calcium channels (VSCCs) are upregulated in the spinal cord in chronic pain states and, x-conotoxins specific for N- type VSCCs are potent analgesics (31). Sodium channel inhibitors, and a- and v-conotoxins, which target the nicotinic acetylcholine receptor and the NET respectively, have also been implicated as analgesic agents (30). Vc1.1, an a-conotoxin that is active against the a9a10 nicotinic acetylcholine receptor (nachr), has been proposed to act against that target to produce analgesia (32, 33), although a recent study based on differential activity of post-translationally modified analogs of Vc1.1 suggested that a9a10 nachrs may not be the primary target of Vc1.1 for the treatment of neuropathic pain (34). This finding suggests that the molecular targets for pain therapy with conotoxins may be even greater than previously thought. Future applications of conotoxins may extend to a range of other disease states: sodium channel inhibitors have been implicated as treatments for stroke and epilepsy and drugs that inhibit the NET have antidepressant applications. STRUCTURAL ANALYSIS Methods for Determining Structures of Conotoxins Conotoxins are ideally suited for structural analysis with NMR spectroscopy because of their small size and high solubility and stability. A recent report (35) noted that of the 84 entries for conotoxins that had been deposited in the Protein Data Bank (PDB), more than 90% (79/84) of the structures were solved by NMR spectroscopy. The prevalence of NMR structures compared to X-ray structures for conotoxins differs significantly from the ratio of uses of the two techniques for all protein structures deposited in the PDB, which is about 17% NMR relative to 83% X-ray. This distinction is a reflection of the size of conotoxins, which makes them amenable to NMR, in combination with a general difficulty in finding crystallisation conditions for small disulfide-rich peptides. The structures of conotoxins determined with both X-ray crystallography and NMR are generally very similar (36, 37) as exemplified for conkunitzin-s1 (36, 38) andgi(37, 39). However, in

3 146 DALY AND CRAIK Figure 1. Selected conopeptide sequences. The sequences of contulakin G (5), MrIA (4), PnIA (25), MVIIA (27), and RXIA (10, 11) are given with the disulfide connectivities highlighted on the sequences. These peptides have been isolated from different Conus species and an example of one species, Conus marmoreus, from which MrIA is isolated, is shown on the right of the figure. a recent study on conantokins, although the X-ray and NMR structures were similar, the crystal structure revealed a metal dependent dimerization that was not observed in the NMR structure (40). X-ray crystallography has played a vital role in determining the structures of conotoxins in complex with their binding partners. In particular, complexes of conotoxins with the acetylcholine binding protein (AChBP) have provided significant advances in the field (41, 42). The AChBP is a soluble protein isolated from several non-conus snail species that has high homology to the extracellular portion of the nicotinic acetylcholine receptor and makes an excellent model for probing conotoxin binding. In general, the structures confirm the rigidity of conotoxins because the structures are similar in the unbound and bound states (41, 43). Additional studies utilizing molecular modeling techniques have also provided information about the active conformation of conotoxins (44) but this review focuses on experimental techniques. Structural Folds The sequence diversity of conotoxins is reflected in the diversity of three-dimensional structures that have been determined, as highlighted by the selected examples in Fig. 2. The disulfide connectivity also plays an important role in defining structures. For example, despite having a similar framework of Cys residues, the different disulfide connectivities of the a- and v-conotoxins result in vastly different structures, with the v-conotoxins dominated by b-sheet structure and the a-conotoxins dominated by helical structure. By contrast, there appears to be some particularly stable structural motifs that recur in different conotoxin families. For example, MVIIA and RXIA have significantly different sequences, including a different number of disulfide bonds but the Figure 2. Conotoxin structures. The three-dimensional structures of MrIA (PDB code 3ew4), PnIA (PDB code 1pen), MVIIA (PDB code 1mvj), and RXIA (PDB code 1p4l) are shown with the b-strands shown as arrows and the helices as thickened ribbons. The disulfide bonds are shown in ball and stick format. three-dimensional structures both contain a cystine knot motif (45) where two disulfide bonds and their backbone segments form a ring through which a third disulfide bond threads. The cystine knot appears to be a particularly important motif, with a recent analysis revealing that this topology is found in nearly 40% of known cystine-rich peptide domains (46). Although a range of conotoxins display cystine knot motifs, some local structural and dynamic differences have been observed between cystine knot toxins that may be responsible for functional differences. For example, despite the overall topology of MVIIA and MrVIB being similar, there are distinct differences in the flexibility of the structures. Both molecules comprise four inter-cysteine loops and for MrVIB one of these loops (loop 2) is disordered, in contrast to MVIIA, which is well-defined over the whole molecule. A functional difference between these topologically similar peptides is that they target different ion channels (27, 47). In addition to being a potent sodium channel blocker (47), MrVIB is active at molluscan calcium channels (48), and the disorder in loop 2 may account for this cross-channel activity (49). This analysis highlights one of the strengths of NMR, namely that the ensemble of structures is potentially able to give an insight into regions of flexibility, adding another dimension of information. Further information on structural dynamics can be obtained through NMR relaxation studies such as those done on MVIIA (50, 51). These studies highlighted regions undergoing conformational exchange that may have implications for binding to the biological target.

4 STRUCTURAL STUDIES OF CONOTOXINS 147 CONOTOXINS IN COMPLEXES Although determination of the structure of conotoxins bound to their target receptors is the ultimate goal in structural analyses of conotoxins, the receptors are generally large and membrane-bound making them difficult for structural studies. However, for the a-conotoxins, the isolation and X-ray structure determination of the AChBP, a homolog of the acetylcholine receptor ligand-binding domain, has generated new insights into the binding of a-conotoxins (41, 42, 52). Recently, saturation transfer difference NMR studies have also been used to examine a-conotoxins binding to the ACBP, thus highlighting the value of this protein for both crystal and solution state studies (53). In general, a-conotoxins are buried deep within the ligand binding site and do not significantly change conformation, relative to their isolated solution structure, upon binding (41). Furthermore, the binding of a-conotoxins is dominated by hydrophobic interactions. Figure 3 shows the crystal structure of the AChBP in complex with ImI and a comparison of the solution and crystal structures of ImI is also given. It is clear from this analysis that the overall fold does not vary significantly in the solution and crystal states. In the ImI complex, Arg7 and Trp10 play key roles in the interaction with the ligand-binding site but different interactions are observed in the crystal structure of a PnIA analog (41) and a novel conotoxin TxIA (54) in complex with the AChBP. For instance, TxIA shows a 208 backbone tilt relative to other conotoxin complexes. This difference is the result of a salt bridge not present in the other complexes. Analysis of a- conotoxins bound to the AChBP is providing subtype selective information about binding of particular conotoxins, which may facilitate the design of subtype selective conotoxins. In general, the findings from studies of conotoxins in complex with the ACBP have confirmed an early prediction that conotoxins can be regarded as a stable scaffold for the display of amino acid side chains in defined positions (55). OXIDATIVE FOLDING TO ACHIEVE THE THREE- DIMENSIONAL STRUCTURES OF CONOTOXINS Formation of Disulfide Bonds The mechanism by which conotoxins form their well-defined three-dimensional structures is of significant interest, particularly as it will ultimately have a bearing on their use as drugs. Discerning their folding pathways may facilitate the design of modified versions with enhanced activity or specificity and may also lead to higher yields of the correct product, which is important for manufacture of drug products. A range of studies have been performed, including studies of mechanisms of in vitro folding both chemically and using recombinant expression and analysis of the biosynthesis of conotoxins in the venom duct (56). Although the study of the in vivo biosynthesis of conotoxins is in its early stages, a large number of in vitro folding studies have been performed and a recent review summarized these results (56). On the basis of studies on GI and MVIIA it appears that early folding events are determined by the cysteine spacing whereas the production of the final natively folded peptide involves noncovalent interactions among noncysteine residues (56). Studies are now focussing on how conotoxins are folded in vivo with further characterization of the Conus protein disulfide isomerase (57) likely to shed more light on this process. Structural Differences Between Disulfide Isomers The disulfide connectivity is known to have a significant influence on the overall fold of conotoxins. One of the early examples that exemplified this influence was on a-conotoxin GI (39), where the three possible disulfide connectivities, globular, ribbon, and beads, were synthesized. The native, globular connectivity displayed a well-defined conformation whereas the ribbon and beads had progressively less defined structures. Although progress is being made in understanding the influences of disulfide connectivity on structure and how the in vitro folding of conotoxins occurs, differences still arise that appear anomalous in light of the current evidence. An example of surprising structural differences between disulfide isomers was recently reported for the a-conotoxin BuIA (Fig. 4). Although the vast majority of the literature on native conotoxins represents them as well defined peptides with a range of elements of regular secondary structure, this is not always the case. BuIA does not conform to this convention and in fact significant structural differences in the relative order of the structural integrity of the disulfide isomers is observed compared to other conotoxins (58). The native globular isomer of BuIA, although being the favored isomer during oxidative refolding, has multiple conformations in solution, unlike the case for the majority of native a-conotoxins. The major conformation of the globular isomer has the characteristic a-conotoxin fold (59), but the ribbon isomer displays a single well-defined conformation, in contrast to the nonnative ribbon isomers of other a-conotoxins, which are not particularly well structured (58). Overall these results highlight the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure. This information is potentially important for ongoing efforts to understand the structure activity relationships of a-conotoxins. CONCLUDING REMARKS Determination of the structures of conotoxins has played, and will continue to play, a vital role in understanding the function and therapeutic potential of conotoxins. NMR spectroscopy is the major technique used in determining the structures of the isolated toxins and with the advances that are currently taking place in NMR spectroscopy this technique holds promise for further characterizing the structures of conotoxins. In particular, nano-nmr has allowed structural analyses using only nanomole quantities of native venom peptides (60) and saturation transfer difference methods may be a promising technique for studying

5 148 DALY AND CRAIK Figure 3. Co-crystal structure of a bound conotoxin. The crystal structure of ImI bound to the AChBP (PDB code 2c9t) is shown on the left with the bound structure of ImI compared to the solution structure of ImI on the right (PDB code 1im1). Figure 4. Structural analysis of BuIA. The sequence of BuIA with the ribbon and globular connectivities are shown on the left of the diagram. The three-dimensional structures of the globular (PDB code 2i28) and ribbon (PDB code 2ns3) forms are shown on the right. Although the globular isomer is the native form and generally has a single well-defined conformation for BuIA it is the ribbon isomer which displays a well defined conformation without multiple conformations. binding interactions. Although the use of X-ray crystallography in conotoxin research has been limited this is likely to increase in the future and allow the determination of complexes of conotoxins with target receptors or homologues of such receptors and enhance the understanding of how conotoxins exhibit their selectivity and potency. This information is likely to accelerate the progression of conotoxins into the clinic by aiding in studies aimed at designing more specific and potent analogues. ACKNOWLEDGEMENTS Work in our laboratory on conotoxins is supported by grants from the Australian Research Council and the National Health and Medical Research Council (NHMRC). DJC is an Australian Research Council Professorial Fellow. NLD is an NHMRC Industry Fellow. REFERENCES 1. Olivera, B. M. and Cruz, L. J. (2001) Conotoxins, in retrospect. Toxicon 39, Adams, D. J., Alewood, P. F., Craik, D. J., Drinkwater, R. D., and Lewis, R. J. (1999) Conotoxins and their potential pharmaceutical applications. Drug Dev. Res. 46, Miljanich, G. P. (2004) Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr. Med. Chem. 11, Sharpe, I. A., Gehrmann, J., Loughnan, M. L., Thomas, L., Adams, D. A., Atkins, A., Palant, E., Craik, D. J., Adams, D. J., Alewood, P. F., and Lewis, R. J. (2001) Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter. Nat. Neurosci. 4, Craig, A. G., Norberg, T., Griffin, D., Hoeger, C., Akhtar, M., Schmidt, K., Low, W., Dykert, J., Richelson, E., Navarro, V., Mazella, J., Watkins, M., Hillyard, D., Imperial, J., Cruz, L. J., and Olivera, B. M. (1999) Contulakin-G, an O-glycosylated invertebrate neurotensin. J. Biol. Chem. 274, Terlau, H. and Olivera, B. M. (2004) Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol. Rev. 84,

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