Invited Review - ACS Publications - American Chemical Society

JULYIAUGUST 1993 VOLUME 6,NUMBER 4 0 Copyright 1993 by the American Chemical Society

Invited Review Three-Dimensional Structures of Neurotoxins and Cardiotoxins Bernard Rees* and Alexandrine Bilwes UPR de Biologie Structurale, Institut de Biologie Moldculaire et Cellulaire du CNRS, 15 rue Rend Descartes, F-67084 Strasbourg Cedex, France Received March 9, 1993

Animal venoms are complex mixtures containing a number of proteins or peptides with different properties. These components differ from one animal species to another in their structure and in the target at which they are aimed. Some are found in many venoms, while others belong to only one animal family. Among the most widespread and most potent are the neurotoxins, which interfere with nervous transmission. Phospholipases, the enzymes that disrupt the phospholipids of nervous or muscular membranes, are even more universally found in all kinds of animal venoms. Snake venoms also contain myotoxins which cause local necrosis of muscular tissues by acting on receptors of the muscular membranes (1). Crotalidae snakes use toxins acting on blood coagulation (2);cobras and ringhals contain cardiotoxins which cause heart paralysis (3). Hemolytic toxins are present in many insects, the best documented being melittin, the principal toxic component of bee venom (4). This review will be concerned only with neurotoxins and toxins structurally related to them, such as cardiotoxins, which have a 3D1 structure very similar to that of postsynaptic snake neurotoxins. Neurotoxins are toxins that interfere with the transmission of nervous impulse by modifying the function of some particular type of channel in the nerve or muscle membrane. This definition clearly encompasses a vast 'Abbreviations: 20,two-dimensional, 3D, three-dimemionat; AChR, awtylcholinereceptq AaH,Androctonucl awtralisHector;BPTI,bovine pancreatic trypain inhibitor; NOE,nuclear Overhauser effect; ODMR, optical detection of magnetic resonance; PLA2, phospholipase A*;WGA, wheat germ agglutinin.

number of toxins of animal, plant, or bacterial origin. We will, however, limit ourselves to toxins extracted from animal venoms. Plant or bacterial toxins are different worlds, even if some of them are related to animal toxins, in particular through sharing a common receptor. An example is the acetylcholine receptor (AChR),recognized by both the alkaloid curare and the postsynaptic snake neurotoxins. Neurotoxins are very important tools for the study of the different types of channels in excitable membranes and, thus, of the mechanisms involved in the transmission of nervous impulse. Some of them are very selective.They have enabled rapid progress in the identification and characterization of new membrane receptors. A great number of studies are therefore devoted to them. They are mostly proteins or polypeptides. Their primary structure, i.e., the amino acid sequence, is essential information, especially when the sequences of a number of related toxins are known and can be aligned, thus revealing the conserved or type-conserved (Le., always hydrophobic or hydrophilic, or acidic or basic) residues: such residues are usually important, either to stabilize the three-dimensional structure (the most important case is that of cysteine side chains forming disulfide bridges) or for the function of the protein. This knowledge is however not sufficient for a mechanistic study at an atomic level. This demands the 3D structure as a prerequisite, because the interactions depend on the spatial distribution of the amino acids,which cannot be deduced unambiguously from the primary sequence.

0893-228~/9312706-0385$04.00/0 0 1993 American Chemical Society

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386 Chem. Res. ToxicoZ., VoZ. 6, No. 4, 1993

facilitatory toxins

1

I I

Y

nervous impulse

0

cholinesterase

Figure 1. A schematic view of the neuromuscular junction. Different snake toxins can interfere with the transmission of the nervous impulse a t this level by perturbing the emission of the neurotransmitter acetylcholine, blocking its receptor, or preventing its hydrolysis. (After reference 183).

As a consequence, many efforts have been made to crystallize toxins and determine their X-ray structure. In the last years, two-dimensional (2D) proton NMR, which enables the determination of the structure in solution, has also become an efficienttool, especiallyas many toxins are small proteins quite amenable to NMR analysis. Both methods require a relatively large amount of material, and this has often been a problem, for which genetic engineering techniques, by cloning and expressing the DNAs encoding the toxins, provide a solution in an increasing number of cases. This approach presents the additional advantage of enabling site-directed mutagenesis, thus opening new perspectivesfor structure/function studies (5). X-rays and NMR have now yielded a number of precise 3D structures of animal toxins. They provide a solid basis for critical examination of interaction mechanisms suggested by different chemical or biological studies. These toxin structures are the subject of this review. The targets of these toxins, in most cases ion-channel proteins, are known at a much less detailed level. Many useful, but partial, information has been provided by different physical and chemical methods, such as fluorescence or chemical cross-linking. A more global view, albeit at low resolution, may be given by electron microscopy. This is well suited for the study of the quaternary structure, i.e., the assembly of different subunits. But the structural details, necessary for an understanding of the interaction mechanisms, can be given only by X-ray diffraction. No X-ray structure of an ion channel is known at present. More than the size of the molecules [the five subunits of the nicotinic AChR have molecular masses ranging from 40 to 65 kDa (6),which is not much more than the average protein investigated by X-rays in recent years], the difficulty is that of isolation and crystallization. Membrane proteins are extremely difficult to crystallize, because of their large external hydrophobic surface. Nevertheless, methods using detergents exist and have been successfully used in other cases (7).Structures of ionic channels or ionic-channel proteins, and of their

complexes with agonists or antagonists, may well become available in the future.

Snake Toxins The most widely studied animal toxins are, by far, those isolated from the venoms of snakes. This is due, in part, to their high toxicity, and therefore to the necessity to derive efficientvaccinesagainstthem. But the main reason is their practical importance as probes for the study of nervous and muscular mechanisms and as biochemical reagents. They are characterized by an extreme diversity between the numerous species of venomous snakes around the world and even within the same venom. Four families among venomous snakes are generally distinguished: the Elapidae (cobras, kraits, ringhals, mambas, coral snakes), the Viperidae (crotals and vipers), the Hydrophiidae (sea snakes), and the Colubridae, the largest family,in which, however, only a minority of species are venomous. The composition of snake venoms differs very much from one snake family to the next, and even within a given family. For example, the Elapidae family contains 46 different snake species, but only the species Naja and Hemachatus secrete cardiotoxins, and only the speciesDendroaspis has fasciculins. The neurotoxins are present in most venoms but are of different types depending on the type of membrane receptors or channels upon which they act (Figure 1). PostsynapticNeurotoxins. Postsynaptic neurotoxins are also called a-neurotoxins or curaremimetic toxins, because their action is similar to the alkaloid poison d-tubocurarine (curare). They bind to the nicotinic AChR on the postsynaptic membrane of the neuromuscular junction and thus prevent the binding of the neurotransmitter acetylcholine. This results in flaccid paralysis. These toxins are found in proteroglyphous snakes (the fangs of these snakes are fixed at the front of the jaw, in contrast to Viperidae) of the Elapidae and Hydrophiidae families. There are two classes of a-neurotoxins. Short neurotoxins are 60-62 residues long and contain 4 disulfide

Invited Reviews

Chem. Res. Toxicol., Vol. 6, No. 4, 1993 387

111

N -term

\

’

Figure 2. T h e topology of short and long snake a-neurotoxins: erabutoxin b (17)(left) and a-cobratoxin (19)(right). @-Strandsare symbolized by arrows. Disulfide bonds are represented by thick lines. These drawings and those of other figures were made using the program MOLSCRIPT (184). T h e atomic coordinates were taken from the Protein Data Bank (see Table I).

bridges. Long neurotoxins have 70-74 amino acids and 5 disulfide bridges. Many studies have been devoted to both short and long neurotoxins, and more than 100 primary sequences are available. They show extensive sequencehomology within the whole family. This, together with secondary structure predictions and the similarity of the CD spectra (8-IO), indicates highly similar spatial structures. For a review on a-neurotoxins, see ref 11or 12. Several crystal structures of neurotoxins are known, some at high resolution. Four independent crystal structures have been published for the same, or nearly the same, short neurotoxin: neurotoxins b and a, isolated from the Philippines sea snake Laticauda semifasciata (13,14), proved to be identical to erabutoxins b and a from the and toxin b was found Japanese L. semifasciata (15,16), to differ from a by only one residue. Erabutoxin b was refined very carefully at the unusually high resolution of 1.4 A. It was even found that the crystallized molecules could adopt different conformations at several places of the molecular structure, and these were characterized and refined independently. At the end of the refinement, the crystallographicR-factor (the relative discrepancybetween observed and calculated X-ray amplitudes) was 18%for all data between 10 and 1.4 A (17). Another crystal form has been solved and refined recently, to 1.7-A resolution (18). Concerningthe long neurotoxins,a-cobratoxin from Naja naja siamensis (19) and a-bungarotoxin from Bungarus multicinctus (20)have been crystallized and submitted to X-ray analysis. a-Bungarotoxin was refined at 2.5-A resolution to an R factor of 24% (21) and a-cobratoxin at 2.4-A resolution to an R factor of 19.5% (22). A further refinement of the structure of a-bungarotoxin was carried out after correction of several errors in the primary sequence (R = 25 % for all X-ray reflexions with an intensity larger than 2 standard deviations, in the 7-2.5-A resolution range) (23). The a-neurotoxins are rather flat molecules. The dimensions of erabutoxin without the side chains are 32 X 24 X 14 A3. Figure 2 shows a schematic view of the secondary structure of short and long neurotoxins. The

Table I. Three-Dimensional Structures Deposited in the Brookhaven Protein Data Bank. species

resolution(&

protein

code

ref

erabutoxin b erabutoxin a neurotoxin bb a-cobratoxin a-bungarotoxin cardiotoxin Vn4

3EBX 5EBX lNXB lCTX 2ABX lCDT

1.4 2.0 1.4 2.8 2.5 2.5

14 13 11 16 18 46

lDTX a-dendrotoxin phospholipase A2 1PP2

2.2 2.5

96 84

variant-3

1SN3

1.8

104

ATX Ia

SH I

lATX 2SH1

NMR NMR

139 140

melittin

2MLT

2.0

phospholipase A2 1BP2 trypsin inhibitor 4PTI 7WGA agglutinin

2.1 1.5 2.0

snakes

L. semifasciata L. semifasciata L. semifasciata N . naja siamensis B. multicinctus N . mossambica mossambica D. angusticeps Crotalus atrox scorpions C. sculpturatus Ewing sea anemones Anemonia sulcata Stichodactyla helianthus honey bee structurally related compounds bovine pancreas bovine pancreas wheat germ

83 93 77

a The toxin or protein structures discussed in this review, whose atomic coordinates have been deposited in the Protein Data Bank (PDB) (182), are given with their PDB code number. bProbably identical to erabutoxin b.

four disulfide bridges of short neurotoxins cluster in the central part of the molecule. This forms a compact core from which three large but unequal loops emerge like three fingers. The secondary structure is exclusively of @ type: a three-stranded antiparallel @-sheetencompasses loop I1 and one strand of loop 111, while a second shorter antiparallel @-sheetis formed by part of loop I. The fourth disulfide bridge between cysteines 62 and 682strengthens the C-terminal part of the molecule, which forms a short loop held on top of the molecular core by hydrogen-bond interactions with the N-terminal part. The long neuro2Throughout this review, the amino acids of “three-finger”snake toxins are numbered according to Chart I.

388 Chem. Res. Toxicol., Vol. 6, No. 4, 1993

Rees and Bilwes

Chart I. Consensus Sequences in Snake Toxins: Long a-Neurotoxins, Short a-Neurotoxins, and Cardiotoxinsa

10

20

30

40

50

60

70

r-hgaro

RTCLI***SPSSTPQTCPNGQDICFLKAQCDKFCSIRGPVIEQGCVATCPQFRSNYRSLLCCTT*DNCNH

fasciculin

TMCYSHTTTSRAILTNC**GENSCYRKSRRHP****PKMVLGRGC**GCPPGDDY*LEVKCCTSPDKCNY

RICYIHKASLPRATKTC**VENTCYKMFIRTQ****REYSIERGC**GCPTA~P*YQTECCKG*DRCNK a Symbols used are as follows: @ for Lys or Arg; f for Lys, Arg, His, Asp, Asn, Glu, or Gln; X for Ser or Thr;J. for aromatic;6 for hydrophobic; and X for non-conservedresidues. Deletions are representedby dots. Residuescommon to the three families are boxed. The disulfide bridges are indicated by horizontal lines. The sequences of K-bungarotoxin, fasciculin F7, and calciseptin are shown for comparison (92). dcisqtin

toxins retain this general conformation, but loop I1is longer and its extremity is secured by an additional disulfide bridge. In cobratoxin this extremity contains a one-turn helix (24). Furthermore, an additional tail extends the C-terminal loop. Structural information in solution has been obtained from numerous proton NMR studies, on both short and long neurotoxins. Short neurotoxins include the already mentioned erabutoxins from L. semifasciata (25), neurotoxins I and I1 from Naja mossambica mossambica (26,27),and cobrotoxin from Naja naja atra (28). Among the long neurotoxins are toxin LS I11from L. semifasciata (29),a-bungarotoxin from B. multicinctus (30,31),toxin B from Naja naja (30),and a-cobratoxin from N . naja siamensis (32). The most complete and detailed information is given by 2D proton NMR, where nuclear Overhauser effect (NOE) data are directly related to short proton-proton distances. Using those as structural restraints, together with some torsion angles derived from coupling constants, it is possible to obtain an ensemble of possible 3D structures which can be refined by simulated annealing techniques. A complete description of the hydrogen-bond structure was obtained in this way for a short neurotoxin from Dendroaspis polyepis polyepis (331, for cobrotoxin (34),for toxin a from Naja nigricollis (351, for a-cobratoxin (24, 36) and for a-bungarotoxin (37). The results of these studies are in agreement with the X-ray structures. Only one real discrepancy has been found: in the case of a-bungarotoxin (23,37),NOES were observed in the NMR spectra between Trp 29 and Val 40, indicating a short distance between the side chains of these two residues, while in the X-ray structure they are on opposite sides of the @-sheet. Trp 29 is absolutely conserved in all Elapidae a-neurotoxins; therefore, its orientation is probably crucial for biological activity (31). The triple-stranded @-sheetextends further in the solution structure than it does in the crystal. On the whole, the solution structure of a-bungarotoxin resembles more the structure of a-cobratoxin where the observed differences between NMR and X-ray structures are more subtle. They concern essentially the conformation of the most flexible parts of the molecule, namely, the C-terminal extremity and the tip of loop I1 (24). A case like that of a-bungarotoxin is quite exceptional. Even though a protein may well be flexible enough to adopt different equilibrium conformations in a crystal and in solution, one should not forget that, due to the large quantity of solvent present in protein crystals (often more than 50% of the volume), most interactions between protein and solvent are already present in the crystal.

Another interesting question is that of a possible dependence of the conformation on the pH. This may be relevant to the activity since the toxin is stored at low pH in the venom gland, but acts at physiological pH when injected. There is no evidence as yet for pH-dependent conformational changes (32,36). A structural analysis by 2D NMR has been undertaken for cobratoxin at pH 3.2 and 7, but only the results at low pH have been published so far (24). NMR can provide information on the pK of ionizable side chains, by analyzing the pH dependence of proton chemical shifts. This may be related to the environments of these residues (29). Amide protons involved in H-bonds exchange slowly. Their exchange rate depends on the rigidity of the structure. It has thus been shown that long neurotoxins, and particularly 8-bungarotoxin,have a more rigid core than short neurotoxins (30). This has been attributed to the additional tail, which contains several hydrophobic residues. It may be correlated with the mode of interaction with the AChR: the dissociation constants are very similar for short and long neurotoxins [(6-8 X MI, but short neurotoxins associate and dissociate 5-9 times faster than long neurotoxins (38). The neuromuscular blockade produced by the long neurotoxins is thus more irreversiblethan in the case of short neurotoxins. Once the 3D structure of a toxin is known, the important question is of course its relation to the function, and first of all the delimitation of the “toxic site”, in other words of the region(s) in contact with the receptor. The best way to do this is to modify or mutate some possibly important residues and to measure the change of affinity with the receptor. Caution is required for the interpretation, since some residues may be important not because they are in contact with the receptor, but because they maintain the architecture of the molecule. An obvious example is provided by the cysteines forming disulfide bridges. In the case of short neurotoxins, potentially important residues may be identified by their invariance. Those involved in maintaining the conformation can be deduced by comparison with the structurally homologous but functionally different cardiotoxins (see below): several residues are thus conserved or at least type-invariant in both families of toxins, including the 8 half-cystines. They are shown in boxes in the sequence alignment of Chart I. The remaining residues, invariant in neurotoxins only, are the “functional invariants”. They form the toxic site, as has been verified for some of them by chemical modification: modification of Trp 29, Lys 27, or Lys 53 causes a large increase in the dissociation constant from the cholinergic receptor (39). The functionally invariant

Inuited Reviews

Figure 3. Comparison of the toxic site of a short a-neurotoxin (left) with d-tubocurarine (right). Positivelycharged groups are shown by $, oxygen atoms by black dots, and hydrophobic residues by dotted circles. Arg 33, Ile 36,and Glu 38 become Arg 37, Ile 40, and Glu 42, respectively, in the nomenclature used in the present review. Reprinted from Toxicon, Vol. 20, MBnez et al., Comparison of the “toxic” and antigenic regions in toxin a isolated from Naja nigricollis venom, pp 95-103, with kind permission from Pergamon Press Ltd.,Headington Hill Hall, Oxford OX3 OBW,U.K.

residues are mainly localized in loops I1 and I11 and form a continuous region in space. Two features, observed in a number of agonists and antagonists of the nicotinic AChR, including acetylcholine, nicotine, strychnine,etc., are generallyconsidered essential for the recognition of the nicotinic AChR a positive charge and a hydrogen-bond acceptor, separated by a distance of 5.9 A. These were observed not only in acetylcholineitself (the quaternary ammonium cation and the carbonyl oxygen) but also in a number of other agonists or antagonists of AChR, like nicotine, the alkaloid cystine, strychnine, etc. (40). In erabutoxin, these requirements could be met by the guanidinium group of Arg 37 and a carboxylate oxygen of Asp 31. Both residues are among the functional invariants and occupy the extremity of loop 11. The two groups would indeed be separated by 5.9 A, if the amino acid side chains were linked by a hydrogen bond (41). Such a pairing is however not observed either in the crystal nor in the NMR structures, probably because in both cases the ions are surrounded by water molecules. But they could easily be brought to interact in a more hydrophobic environment. Tsemoglou et al. (41)therefore proposed a model in which Arg 37 and Asp 31 block the acetylcholine binding site on the receptor, while Trp 29 participates in a hydrophobic association and the conserved lysine residues give rise to ion pairing with the receptor. Similarities have also been observed between the functionally invariant residues of loop I1and the structure of d-tubocurarine. The distance between the side chains of Lys 27 and Arg 37 is only slightly larger than the distance between the two quaternary nitrogens of tubocurarine, and in both cases these positive charges are separated by a hydrophobic or aromatic patch. There are also electronegative groups (oxygen atoms) in similar positions (Figure 3) (42). Experimental evidence of the importance of loop I1 for toxicity has been obtained by Juillerat et al. (43), who showed that a synthetic peptide formed by 33 residues of an a-neurotoxin from Naja naja philippensis was highly active in binding isolated AChR. This peptide contained essentially the residues of loop I1 and of the first half of loop I11 (assuminga structure similar to that of erabutoxin), cross-linked by only one disulfide bridge. Although the

Chem. Res. Toxicol., Vol. 6, No.4,1993 389

Kd was much larger than for the complete toxin (2.2 X lo-’ M instead of 3 X 10-lOM),it was still an order of magnitude smaller than that of acetylcholine itself. A detailed discussion of the mechanisms involved in the binding of neurotoxins on the AChR, based on the high-resolution structure of erabutoxin, has been given by Low and Corfield (44). These authors emphasized the importance of a hydrophobic cleft in the middle of loop 111, which they call the “Trp cleft” because it could accommodate a tryptophan side chain of the receptor. This portion of the toxin is characterized in the crystal structure by a higher thermal-motion factor, indicating increased flexibility. They proposed a mechanism of toxin-receptor binding that first involves Coulombian (ion-pair) interactions with Asp 31,Arg 37 (the two of them mimicking acetylcholine), Lys 27, and Lys 53. The role of these charged side chains is to correctlyorient the toxin molecule at the receptor surface. Coulombian interactions are required at this initial stage because they operate at longer range than van der Waals forces. One of the Trp residues of the receptor would then be locked up in the hydrophobic Trp cleft. This could explain the higher affinity of the neurotoxins compared to acetylcholine. It is also consistent with a study by Trp phosphorescence and optical detection of magnetic resonance (ODMR),which indicated an increased hydrophobic Trp environment in synthetic peptides reproducing the binding-site regions of the AChR, upon binding on a-cobratoxin (45). All details of the interaction with the nicotinic AChR at the neuromuscular junction are not yet known. The main reason is that the detailed structure of the receptor is not available. We know it is a pentamer of the type azOy6, with a total molecular mass of about 250 kDa. All subunits have been completely sequenced in several organisms, and they show extensive homology between themselves. Their order around the axis of the channel is a-y-a-8-6. Each a subunit contains a binding site for acetylcholine, but the two binding sites have different affinities. The two sites need to be occupied simultaneously by acetylcholine for the ion channel to open (for a recent review, see ref 46). The 3D structure is known at a low-resolution level from electron microscopy (47): the five subunits are arranged symmetrically around an axis normal to the membrane. The external diameter is about 80 A and the length along the axis about 140A, with 35-40 A penetrating the cytoplasm and 70 A protruding out on the synaptic side. The channel is like a funnel with a diameter of 25-30 A on the synaptic side which becomes much narrower on the other side. Snake neurotoxins also interact primarily with the a subunits. Electron microscopy at 20-Aresolution showed two a-bungarotoxin molecules making extensive surface contacts with the AChR receptor (6). The resolution is sufficient to exclude the possibility that the toxin acts by physically blocking the entrance to the central cavity. Although more than one sequence segment of the a subunit seems to be implicated in the binding of the ligand (481, the essential role of a peptide of about 20 residues has been demonstrated. It is centered on two conserved cysteines, Cys 192 and Cys 193. Even though occupying adjacent positions, the two cysteines are cross-linked by a disulfide bridge which can be reduced under mild conditions (49). This stretch contains most residues essential for binding not only the neurotoxins and acetylcholine, but also tubocurarine and nicotine (48). There are several conserved tyrosine and tryptophan residues.


It has been suggested that the ir electrons of the aromatic rings help stabilizing the positive charge of the quaternary ammonium of acetylcholine. Such interactions have been studied on a model system composed of acetylcholine and a synthetic receptor comprising primarily aromatic rings (50). However, the quaternary ammonium groups form principally ion pairs with Asp or Glu side chains of the receptor. Cross-linking experiments have shown that negative subsites of the 6 subunit are at less than 9 A from the cysteines of the binding site (51). This means that one binding site is located at the edge of one of the a subunits, at the a-6 interface. The other is probably at the a-y interface of the other a subunit. This is consistent with results obtained through photolabeling with d[3Hltubocurarine (52). Since the a-y and the a-6 interfaces are not identical, it accounts for the two binding sites not having the same affinity for acetylcholine or d-tubocurarine (53). Cross-linking of a-conotoxin (another antagonist of the AChR, see below) showed, even more surprisingly, that the a subunits were less efficiently cross-linked than the other subunits (54). This could mean, not only that the binding sites are at the interface of subunits, but that the toxin groups which were cross-linked (e.g., a lysine amino group) are facing away from the a subunit. Another possible interpretation is that a-conotoxin has additional binding sites. It has been shown that the binding sites of neurotoxin and of acetylcholine overlap but are not identical. This may explain the resistance of snakes to a-neurotoxins: in snake AChRs, several amino acids of the binding region are changed, in particular one tyrosine into a nonaromatic residue. These mutations result in a much lower affinity for neurotoxins, witout affecting the affinity for acetylcholine (55). Toxins Acting on Other Cholinergic Receptors. Neuronal neurotoxins, also called K-neurotoxins, are postsynaptic neurotoxins that block nicotinic AChRs of the peripheral nervous system, rather than the AChRs of the neuromuscular junction (56). The solution structure of K-bungarotoxinhas been determined by 2D NMR (57). This toxin exhibits a considerable sequence homologywith a-bungarotoxin, but also a few important differences at key positions. For example, Trp 29 discussed above is replaced by a Gln (but the side chain remains on the same side of the @-sheetas in the solution structure of a-bungarotoxin). The C-terminal tail is missing. NOE connectivities indicate a three-stranded antiparallel @-sheet practically identical to that of a-bungarotoxin. Additional NOES are consistent with a dimeric association. This causes the formation of a six-stranded antiparallel @-sheet extending over two molecules. A similar situation has been observed, albeit only in the crystal form, for a-bungarotoxin (21) and a-cobratoxin (221, and also in the case of a cardiotoxin (58). To get a better insight in the structural basis of the different activities of a-and K-neurotoxins,a precise X-ray structure would be required. K-Bungarotoxin has been crystallized with the scope of determining the structure. Preliminary crystal data have been published (59). A different type of AChRs in the central and peripheral nervous system is called muscarinic because these receptors are not sensitive to nicotine but to the mushroom toxin muscarine. Two toxins that affect these receptors have been characterized in the venom of green mambas (60). Their length is similar to that of short a-neurotoxins, and

Rees and Bilwes

they too have four disulfide bridges. Indeed, the published primary sequence of one of them shows several homologies with erabutoxin, including identically positioned cysteines (61). A similar 3D structure is therefore expected. Cardiotoxins. Cardiotoxins are basic proteins found only in the venom of the cobras (Naja and Hemachatus), where they represent about half of the total protein content. They are less toxic than neurotoxins. As their name indicates, they cause heart failure, but they are also known to depolarize muscular membranes and cause contraction of smooth and skeletal muscles, to depolarize nerve cells, to cause cytolysis and hemolysis, and to prevent platelet aggregation (62). For all these reasons, they are called not only cardiotoxins but also cobramines, cytotoxins, cytolysins, direct lytic factors, membranotoxins, or membrane-active polypeptides. This abundant terminology reflects a lack of understanding of what really is the main target of these toxins. Indeed, this is still controversial. No receptor has been unambiguously identified. It is clear, however, that cardiotoxins act in a manner completely different to the postsynaptic neurotoxins. In particular, they have no known effect on acetylcholine receptors. Nevertheless, from a structural point of view, they are closely related to the neurotoxins. Cardiotoxins consist of 60-62 amino acids, reticulated by 4 disulfide bridges. More than 60 primary sequences are available. Their alignment shows a number of conserved residues. Since the length is about the same, and the cysteines occupy the same locations, alignment with short neurotoxins is straightforward. However, as discussed above, if one excepts the cysteines, only a few conserved residues are common to both toxin families (Chart I). Among these are a proline and a glycine which, as usual, play a structural role in stabilizing some particular conformations. The structural similarity with neurotoxins is confirmed by the 3D structure. Only two cardiotoxin crystal structures have been determined: toxin V114 from N . mossambica mossambica (58) and toxin y from N. nigri~ollis.~ The former was refined to an R factor of 22 % using X-ray data to 2.5-A resolution. Toxin y diffracted to high resolution(1.55A),andtheRfactorwas18%. Preliminary crystallographic data have been published for a cardiotoxin from N . naja atra (63). Another toxin from N. mossambica mossambica (CTXIIb)(64) and toxin y from N. nigricollis (65,53)have been submitted to 2D NMR analysis. These structures show the same folding as a-neurotoxins, characterized by two antiparallel @-sheets(Figure 4). The core is almost exactly superimposable on that of erabutoxin or of cobratoxin. This includes the disulfide bridges, the @-sheets,and the C-terminal loop, even for the long neurotoxin if the last 8 residues are omitted (58). The loops, however, show important differences, particularly the long and very flexible loop 11, which is flipped from one side of the molecule to the other, so that the concavity of the molecule is changed (Figure 4c). The crystal structures show the existence of a @-bulgein this loop in the cardiotoxins. A conserved proline at position 40, absent in the neurotoxins, may be important for forcing this conformation. The exchange rates of slowly exchangeable amide protons, as measured by NMR, were found to be considerably faster in cardiotoxin from N . mossambica mossambica than in several neurotoxins. This indicates 3Bilwes et al.,unpublished.

Invited Reviews

Figure 4. Comparison of a short a-neurotoxinwith a cardiotoxin (stereoviews). Residues are numbered as in Chart I. The top two panels show the network of hydrogen bonds between mainchain atoms: [panela (top)] in neurotoxin (17);[panel b (middle)] in cardiotoxin(toxin T ) . ~The regular hydrogen-bond pattern of the &sheet is disrupted in loop I1 by a 8-bulge at residues 34 and 35. [Panel c (bottom)] Superpositionof cardiotoxin (thick line) and neurotoxin (thin line). This is a side view, with loop I in front. Notice the different conformationsof loops I and I1 in the two toxins.

a more flexible structure (26,27). The crystal structure of toxin y clearly shows this flexibility: important conformational differences could be observed between the three independent copies of the molecule, particularly in loops I and II.3 Conformational changes upon binding to a lipidic bilayer, with an increased content of @-structure, were also indicated by a study of three cardiotoxins by infrared spectroscopy (66). According toa classificationbased on the sequence, more specifically on the presence or absence of two proline residues at the tip of loops I (position 12) and I1 (position 37), the two cardiotoxins of known X-ray structure belong


to two different groups (3). Although their CD spectra are also different (65), a comparison of the structures reveals that they exhibit a number of identical features, even at a detailed level (conformations of disulfide bridges, for e ~ a m p l e ) There .~ are, however, important differences in the conformation of loop I, which is essential for the function, as will be discussed below. The most interesting and important differences between a-neurotoxins and cardiotoxins concern the nature of the amino acid side chains and their spatial distribution. Cardiotoxins are rich in hydrophobic and basic amino acids. The hydrophobic residues belong to different strands of the polypeptidic chain but form continuous clusters. The extremity of loop I, in particular, is completely hydrophobic. The basic side chains tend to be concentrated on the border of these clusters. This becomes particularly clear if one considers only the typeconserved residues (Figure 5a). Nothing comparable is observed in neurotoxins, where loop I, for examde. is hydrophilic (Figure 5b),A d the onlyhydrophobic iegion is that of loop I11 discussed above. The existence of external hydrophobic regions puts constraints on the crystal packing, which tends to hide these parts from the solvent. Cardiotoxins are often difficult to crystallizefor this reason. The two cardiotoxins of known crystal structure have found different “solutions” to the problem of hiding the hydrophobic zones, resulting the in unusual packing arrangements. In cardiotoxin VII~, molecules are arranged in a helical way around 6-foldscrew axes. This leaves much empty space, so that the solvent content in the crystals is quite high (58). In toxin y, three molecules pack together in the crystal independent unit. This enables a much tighter packing, which accounts for the higher resolution limit of the X-ray diffraction.3 Clearly, this pattern of hydrophobic and basic residues in the cardiotoxins is functionally important. It is known that cardiotoxins are able to attack and disrupt membranes or to penetrate acidic phospholipid monolayers (67). It can be imagined that the hydrophobic zones, particularly the completely hydrophobic tip of the first loop, penetrate the inner part of a membrane, while basic side chains interact with the acidic heads of the phospholipids (68). Fluorescence measurements on cardiotoxin V I I ~from N . mossambica mossambica have shown that Trp 14 penetrates deeply into a model cardiolipin membrane (69).Can a cardiotoxin completely cross a lipidic bilayer, so that the hydrophilic tip of loop I1would emerge on the cytoplasmic side? The length of the hydrophobic region seems insufficient to completely span the acyl chain region of the lipidic bilayer, even if the side chains of lysine and arginine residues are completely stretched out on both sides, thus maximizing the size of the hydrophobic portion of the cardiotoxin molecule. However, local lipid structures different from a bilayer may need to be considered. In their study of the action of a cardiotoxin on cardiolipin model membranes, Bratenburg et al. (691, found evidence that cardiotoxin was inducing fusion of lipid vesicles, resulting in changes in the lipid structure, with the probable formation of inverted micelles. The hydrophobic and hydrophilic regions of cardiotoxin fit well a model structure of such micelles. The coexistence of an exposed hydrophobic surface and of a cationic zone is a characteristic of a number of proteins or peptides which all exhibit cytolytic activity. Besides cardiotoxins, these include snake myotoxins which cause muscle degeneration ( I ) ,the melittins of bees which cause


Rees and Bilwes

Invited Reviews


Figure 5. The invariant and type-conservedamino acids in [panel a (top left)] cardiotoxin (58);[panel b (lower left)] a-neurotoxin [erabutoxin b [panel c (above)] a scorpion toxin (124). A side chain is represented by colored atomic spheres when it conserves its character in at least 80% of the primary sequences available. The characters considered are as follows: basic (shown in green), acid (red),neutral hydrophilic (blue),and hydrophobic (yellow). The cysteines (disulfide bonds) are in gray. Extended hydrophobic patches are seen in cardiotoxin and in the scorpion toxin. In cardiotoxin the extremity of loop I (on the right side of the molecule) is completely hydrophobic. Basic residues concentrate at the border of the hydrophobic patches. Snake neurotoxin is shown for comparison: its amino acid distribution is completely different. Except for a hydrophobic patch on loop I11 [which has probably a functional role in binding to the AChR ( 4 4 ) ] ,there is no significant accumulation of hydrophobic residues.

(In];

lysis of erythrocytes ( 4 ) , other insect toxins such as cecropinsor sarcotoxins, magainins from amphibians, and several microbial hemolytic toxins (70).The presence of both the cationic and the hydrophobic site seems to be essential for the lytic activity, since no such activity is found in highly basic proteins not possessing a suitable hydrophobic surface (myelic basic proteins, protamines, histones); on the other hand, the lytic activity is lost (but not the ability to bind phospholipids) in modified cardiotoxins where the positive charges of the lysine residues have been removed, while the activity is retained when the same lysines are guanidinated, thus conserving their positive charge (71). Although hemolysis is certainly not the most important effect of cardiotoxins in uiuo, many studies have been devoted to the action on the red blood cells. A problem encountered when studying cell lysis by cardiotoxins is contamination by small quantities of phospholipase A2 (PLA2) with which they synergize. In the absence of contamination, hemolytic activity is strongly reduced and occurs only at relatively high concentration (72,73). In this respect, cardiotoxins differ markedly from melittin, which is able to interact with neutral phospholipids (67) and can therefore rapidly penetrate the outer face of an erythrocyte membrane. The hemolysis of erythrocytes by cardiotoxinis preceded by a rather long nonlytic period, during which the fragility and osmotic sensitivity of the erythrocyte membrane increases. When PLA2 is added during this period, the erythrocytes immediately start to break up (74).At least in this case, cell lysis seems rather due to the hydrolysis of phospholipids by PLA2, facilitated by an alteration of the cell membrane structure caused by cardiotoxin. Toxicity of most cardiotoxins is essentially due to depolarization and contraction of cardiac muscles, which

begins at much lower concentrations. Depolarization of skeletal musclesis also induced. Muscle cell depolarization is often used as an assay for cardiotoxin activity, since it is not too sensitive to PLA2 contamination. The depolarization of the muscular membrane activates the voltagedependent Ca2+channels with subsequent release of Ca2+ from the sarcoplastic reticulum, and therefore muscle contraction. However, it has been shown that contraction can occur even in low-sodiummedia (75),which apparently indicates that membrane depolarization is not an absolute prerequisite. It has been suggested that, rather than to a voltage-dependent effect, the muscular contraction induced by cardiotoxin is due to the activation of an endogenous Ca2+channel (3). The mechanism of the depolarization is still obscure. It could be due to the formation of pores which would allow the free passage of ions (76). The structure of toxin y from N . nigricolzis provides some support t o this hypothe~is:~ three independent molecules associate in the crystal in a porin-like manner, roughly forming a cylinder of about 40-Aexternal diameter and 30-Athickness. The central channel narrows to a few angstroms. Most hydrophobic side chains are directed outward. The association is however very loose, and the presence of positive side chains on the inner side would not favor the passage of positive ions. The formation of a pore by oligomeric association with a protein, such as band 3 protein in the erythrocyte membrane, has also been suggested (3). It is known that cardiotoxinsinteract with phospholipids in natural and synthetic membranes, but only negativelycharged lipids are able to associate (77). Cardiotoxin is in competition with Ca2+ions for this association. The affinity of cardiotoxin for negative lipids is high (& < M), and the stoichiometry is about 7 lipid molecules

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for 1 molecule of cardiotoxin, which is slightly less than the number of positive charges available in the toxin molecule. The importance of loop I, with its completely hydrophobic extremity, has been emphasized for the interaction with phospholipid vesicles, and the affinity increases when an arginine is present at position 5, just before the hydrophobic patch of this loop (68). The main argument against the hypothesis of a phospholipid target i n uiuo is that the cell membranes so far investigated contained only a small amount of negative phospholipids which furthermore were essentially concentrated on the cytoplasmicside,and therefore were not directly accessible. However, it has also been shown that the composition is very dependent on the type of cell (78). We must also bear in mind that the experimentally-determined compositions are mean values and that in some cases local clustering of negative phospholipids is perhaps not excluded. It is therefore not possible to exclude altogether the phospholipids as the main target of cardiotoxins in some particular membranes, although it is not very likely. Another possiblity would be the negative carbohydrates, abundant on external membranes. But they seem to be ruled out, since there is no loss of cardiotoxic activity after treatment of the cells by neuraminidase, which removes outer glucidic parts of the membrane glycoproteins (79). Some proteins of the membrane also contain negatively charged regions. Such anionic regions commonly occur at the boundary with the lipid bilayer, close to the hydrophobic membrane environment. The endogenous Ca2+ channel mentioned above is such a possible protein target (3). Recent cross-linking experiments using radiolabeled cardiotoxin support the hypothesis of a protein target: these experiments suggested that cardiotoxin could bind specifically to a protein of molecular mass 59 kDa from the cockroach heart membrane (80). The problem is further complicated by the heterogeneity of the cardiotoxin family (65). The CD spectra suggested conformational differences, leading to the distinction between two subclasses (82). There is also heterogeneity in the biological properties. Thus, some cardiotoxins belonging to the subgroup called cardiotoxin homologues lack the ability to contract muscles but possess potent lytic activity. This suggests distinct mechanisms for lytic activity and for cardiotoxicity (72). Similar differences between cardiotoxins have been observed concerning the ability to depolarize cell membranes and the capacity of hemolysis (73), or for the fusion of lipid vesicles and hemolysis (82). Finally, no correlation could be found between lethality and depolarizing or lytic activity, in contrast to a-neurotoxins where the lethal potency correlates well with the affinity for the nicotinic AChR (65). Almost as controversial as the identification of a receptor is the location of a functional site in cardiotoxins. The different approaches used to identify this site led to rather contradictory results. Correlations between primary sequence and depolarizing activity led to the conclusion that the functionally important residues were located in all three loops, and particularly in loops I1 and I11 (3). For example, Ser 53, in loop 111, was found to be of particular importance: it is conserved in most cardiotoxins, but in cardiotoxin N. naja atra I it is changed to Asn. This is the only difference with N. naja siamensis CM6 (except for the reverse change-Asn to Ser-at the nonconserved position 52), but the activity is reduced by 1 order of magnitude (73). This method of identification is not of general use because in most cases two cardiotoxin se-

Rees and Bilwes quences differ in more than one place: the observed effect is the resultant of all the individual changes, which makes interpretation difficult. Another, probably more convincing, approach involves mutations or chemical modifications. Oxidation of methionines 27 and 29 (loop 11) results in a dramatic loss of toxicity or muscle contractile activity for several cardiotoxins (83, 84). Chemical modifications of aromatic side chains and amino groups of toxin y from N. nigricollis have shown the importance of loop I (particularly the conserved Lys 15) and to some extent the base of loop 11, for cytotoxicity, cell depolarization, and lethal activity in mice (65,851. By contrast, modifications made in loop I11 and at the tip of loop I1 had no noticeable effect. Other molecular regions may be of importance. There is, for example, a well-conserved sequence, -Cys-Pro-XGly-Lys-Asn-Leu-Cys-,on the side of the molecule opposite to the three loops (residues 17-24). X is mostly Glu or Ala, and in a few cases Lys. In their analysis of the interaction of cardiotoxins with sphingomyelin vesicles, Chien et al. (82) found that a cardiotoxin with X = Glu had a 10-foldhigher ability to induce fusion of the vesicles than another with X = Ala. The authors proposed for this reason that X together with Lys in the same patch would be a site of interaction with the zwitterionic headgroup of these phospholipids. However, the nature of the residue X is not the only difference in the sequences of the two cardiotoxins. The heterogeneity of the cardiotoxins may be one of the reasons for divergent interpretations, since these studies use different cardiotoxins and analyze different effects, which need not necessarily involve the same residues. Hider and Khader (72) suggested the existence of distinct functional sites for cardiotoxicity and for lytic activity: they stressed the importance for cardiotoxicity of the middle of loop 11, which contains methionines 27 and 29, but lytic activity would depend on the hypervariable extremity of loop 11. They even found a correlation between the net charge of the corresponding amino acids and the lytic activity. Fasciculins. These toxins have been found only in the venom of one snake species, Dendroaspis (mamba snakes). They are inhibitors of acetylcholinesterase. This enzyme hydrolyzes the acetylcholine molecules released into synaptic space at the neuromuscular junction and thus contributes to termination of transmission of the nervous impulse. Fasciculins act in synergy with dendrotoxins, which increase the release of acetylcholine (see, below, the presynaptic toxins). In contrast to neurotoxins which block transmission, fasciculins and dendrotoxins increase it beyond control, thus causing a permanent muscular contraction. The crystal structures of two fasciculins have been recently determined by X-ray diffraction (86). These toxins have 61 residues, 4 disulfide bridges, and the same residues as short neurotoxins and cardiotoxins at the sequence-consensus positions (seeChart I). The structural similarity with neurotoxins and cardiotoxins is indeed striking. The molecules have the characteristic threefinger structure, and their core is superimposable on that of short neurotoxins and cardiotoxins. As in the neurotoxin-cardiotoxin comparison, the loops show some conformational differences, but they are closer to cardiotoxin than to neurotoxin, especially loop 11. The structure of fasciculin 1 has been solved to a resolution of 1.9 A ( R = 18% for all reflections in the

Invited Reviews

resolution range 8-1.9 A) (87). Fasciculin 2 has been solved to 2.3 A and is in the process of refinement (86). The molecules differ by only one residue, which is either Tyr or Asn at position 47. Surprisingly, the two structures show a large conformational difference in their first loop, a region far from the substitution. This is certainly due to different packing interactions in the crystal and illustrates the flexibility of the loops. In the case of fasciculins, not only is the receptor clearly identified but its detailed 3D structure is known since the recent publication of the crystal structure of acetylcholinesterase at 2.8-A resolution (88). The catalytic site is well characterized. The next step would logically be the crystallization and structure determination of the toxinenzyme complex, which would enable for the first time direct observation at the atomic level of the interactions between a snake toxin and its receptor and possibly reveal the mechanism of inhibition. Other Toxins with a Probable Three-FingerStructure. The fasciculinsbelong to the so-called“Angusticepstype” family. The toxins of this family, which come mainly from mamba venoms, show sequence homology with a-neurotoxins but are less lethal, probably because they lack some residues essential for neurotoxicity (89). Calciseptin, another member of this family, has been isolated recently from the venom of the black mamba venom. This toxin has been shown to block selectively L-type Ca2+ channels, and to abolish contractions in cardiac and smooth muscle cells (90). It is 60 residues long and has 4 disulfide bridges. Its primary sequence is extensively homologous with that of the fasciculins. Angusticeps-type toxins contain all the “structural” residues (those also present in cardiotoxins-see Chart I); therefore, they are almost certainly all characterized by the same three-finger folding. A number of other toxins isolated from different elapid snake venoms [particularly the egyptian cobras Naja haje (91)l showed the same sequence homology with neurotoxins and cardiotoxins. They are all of low toxicity. For a compilation of the sequences and references, see ref 92.

The “Toxin-Agglutinin” Fold. All the structures discussed up to this point have the same folding: they are small, rather flat proteins, with a tight core secured by four disulfide bridges (the fifth bridge found in long neurotoxins is outside this core), from which three main loops extend like three fingers. The same fold has been found in a protein that apparently bears no relation to snake toxins, namely, wheat germ agglutinin (WGA) (93), a saccharide-binding plant lectin. WGA is a dimer, each protomer consisting of four structurally homologous domains, each with four disulfides and the three-finger fold (94). Since one domain is only 43 residues long, the three loops are smaller than in the snake toxins and too short to form a /3-sheet structure, but the four disulfide bridges are nearly superimposable with those of cardiotoxins or neurotoxins, and the last bridge closes a loop at the C-terminal end, which is of the same length (6 residues) and in a very similar position (Figure 6). The only loose functional relationship between WGA, cardiotoxins, and neurotoxins is that they are all able to attach themselves to some (different) components of cell membranes. Other plant proteins of similar size having this property contain 8 half-cystines at approximately the same position in the sequence as WGA and presumably have the same folding (93). There seems thus to exist a whole class of proteins in widely different organisms,


Figure 6. The “toxin-agglutinin” fold. Schematicrepresentation of the polypeptide backbone and disulfide bonds of one of the wheat germ agglutinin domains and of erabutoxin b. Reprinted with permission from Drenth et al. (1980) J. Biol. Chem. 255, 2652-2655(93).

differing by their function and by their primary sequence, which all exhibit a similar “toxin-agglutinin” fold. Presynaptic Neurotoxins. Phospholipases. Dendrotoxins. Presynaptic neurotoxins are found in the venom of elapid and viperid snakes. They act at the presynaptic level by interfering with the release of acetylcholine or other neurotransmitters. This effect, called P-neurotoxicity, is due to the inhibition of a voltagesensitive K+ channel. The &neurotoxins are a subclass of the PLA2. Neurotoxic as well as nonneurotoxic snake venom phospholipases show extensive sequence homology with PLA2s from mammalian pancreas. It is not yet quite clear whether the enzymatic activity is necessary for the neurotoxic action (95). Besides neurotoxicity, snake venom phospholipases may induce a variety of pathological effects in different animal tissues or organs. They can also be myotoxic, cardiotoxic, or hemolytic and may affect platelet aggregation. To explain these observations, Kini and Evans postulated the existence in the PLAz protein of a “pharmacological site”, distinct from the catalytic site, and able to recognize in vivo a particular “target site” on a target cell. After the specific binding, the PLA2 can hydrolyze neighboring phospholipids, an action which may or may not be necessary for the induction of pharmacological effects (96). However, the mechanisms of the various types of activity and the nature of their interrelations are still not clearly understood (97). Snake venom phospholipases form an extremely vast and diversified family. Neurotoxic phospholipases include, among others, notexin (Australian tiger snake), taipoxin (Oxyuranus scutallatus scutellatus ), and P-bungarotoxin (B. multicinctus) (all from elapid snakes), and caudoxin (Bitis caudalis) and crotoxin (Crotalus durissus terrificus) from viperid snakes (98). All of them have a PLAz-like primary (and presumably tertiary) structure, with the exception of the B chain of 6-bungarotoxin, as discussed below. Some act in amonomeric form. Taipoxin is a ternary complex of three nonidentical subunits which act synergistically, thus conferring on this toxin an extremely high toxicity (99). Crotoxin is made of two subunits, both of them homologous with PLA2, even though the second subunit (crotapotin) is cleaved into three polypeptide units (100). This toxin also has the peculiarity of interfering with neurotransmission at the postsynaptic level. Two other toxins, vipoxin and 0-RTX, isolated from the venom of Russell’s viper, also belong to the PLAz family (101,102). They act on noncholinergic receptors. They were found to inhibit biogenic amine receptors (adrenergic,


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C-term

Figure 7. The phospholipase A2 fold [bovine pancreas PLAz (103)l. The a-helices are labeled A-E.

dopamine, or serotonin receptors). P-RTX is smaller than vipoxin and than the usual PLA2 enzymes, as shown by its lower molecular mass and the presence of only 8 halfcystines. 8-Neurotoxins and PLA2 are proteins of 120-130 amino acids with 6 or 7 disulfide bridges (Figure 7). Sequence comparison (and in particular the position of the cysteines formingdisulfidebridges), secondary-structureprediction, and circular dichroism indicated that the homologous @-neurotoxinsand PLA2 would all have a similar folding and conformation. This was found to be the case for the known X-ray structures of phospholipases both of mammalian origin (103)and from the venom of Crotalidae (104, 105)or elapidae (106).The crystal structure of a @-neurotoxic PLA2, notexin, has been recently published (107). This structure has been refined to an R factor of 16.5% at 2-A resolution. It again showed a striking similarity both to snake venom and to mammalian PLA2 (Figure 8). The main-chain conformation of notexin is even closer to that of bovine PLA2 than to that of rattlesnake PLA2. All 7 disulfide bridges have the same location in notexin and in bovine PLA2, while one is different in the rattlesnake. Several preliminary accounts have been published of X-ray structure determinations of other 8-neurotoxins, namely, 8-bungarotoxin(108), crotoxin (log),and caudoxin (110). Dufton et al. (111,112) pinpointed a certain number of sites in the PLA2 polypeptide chain that appear essential for the neurotoxic activity. This study indicated a toxic site distinct from the protein/lipid interface and the catalytic site of the enzyme. It is essentially located in the region of the two-stranded antiparallel @-sheet(residues 70-100). A study based on the local hydrophobic character indicatesa slightlydifferent region, 8+110, which contains the a-helix E (98)(Figure 7). In most cases, this region is distinctly more hydrophobic in @-neurotoxinsand in neurotoxic phospholipases A2 than in nonneurotoxic phospholipases (98). In notexin, this is also one of the regions where differenceswith the two nonneurotoxicPLA2 structures are most pronounced (107).

Figure 8. The superimposed backbones of the P-neurotoxin notexin (yellow)(Ion,Western rattlesnakevenom PLA2 (green) (104), and bovine pancreatic PLA2 (red) (203).

Among the most studied @-neurotoxinsare the @-bungarotoxins. In these toxins the phospholipase-likedomain, called the A chain, is complemented by a B chain. The A and B chains are connected by a disulfide bond. The B chain is 60 residues long, with three disulfide bridges. It has by itself no neurotoxic nor phospholipase activity. It was shown to be homologous (27-37% sequence homology and 6 common half-cystines) with Kunitz-type proteinase inhibitors such as bovine pancreatic trypsin inhibitor (BPTI) (113) and with several toxins, called facilitatory toxins or dendrotoxins, found in mamba snake venoms (114).These proteins inhibit neither trypsin nor chymotrypsin. Several dendrotoxins and homologues have now been isolated and sequenced. Dendrotoxins come from the venom of mambas, but protease, trypsin, or chymotrypsin inhibitors have been found in the venom of cobras or vipers (115).Besides BPTI, we know the crystal structure of a-dendrotoxin from Dendroaspis angusticeps (116). This structure has been refined to an R factor of 17.5% at 2.2-A resolution. It is nearly superimposable on BPTI, in spite of the completely different origin of the two proteins and their different activity (Figure 9). The structures of the other members of the family are likely to be similar. The structure is rather compact, with a three-stranded antiparallel @-sheetand a two-turn a-helix, held together by a disulfide bridge. The extremities of the longest 0-strands are secured by another disulfide, and a third binds the a-helix, which forms the C-terminus, to a small helical section on the N-terminal side. Dendrotoxins facilitate the release of acetylcholine and of a variety of other neurotransmitters at peripheral and central synapses, an effect opposite to that of P-bungarotoxin which irreversiblyblocks transmitter release [after a transient increase (95)l. However, they can inhibit @-bungarotoxin, indicating that these toxins share a common binding site (117).This is consistent with the hypothesis that the B chain of @-bungarotoxinworks as


Invited Reviews N-term C-term

A59

Figure 10. The folding of long-chain scorpion toxins [here variant-3 (124)l.

Figure 9. a-Dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). [Panel a (top)] fold; [panel b (bottom)] superposition. The thick line represents the Ca backbone of mamba venom dendrotoxin (116),the thin line, that of B P T I (113).

a recognition or binding portion of the toxin to its receptor (118). Both types of toxins work by blocking potassium channels in nerve membranes, but the detailed mechanism is not yet known.

Scorpion Neurotoxins Long-chain Scorpion Toxins. Scorpion toxins are widely used as probes of the sodium channels of excitable membranes (119). They bind with a very high affinity to the voltage-dependentsodium channels where they disturb severalfunctions. They are largely diversified, some being selectively directed toward mammals, others toward insects or crustaceans (see ref 120 for a review). Among the mammal-specific toxins, a distinction is made between a-type toxins found in Old World scorpion species and @-typetoxins found only in the venom of American species. a-Toxins prolong the Na+ inactivation phase of the action potential while @-toxinsaffect the sodium activationphase. The two types of toxins have different binding sites on the sodium channel (ref 121 and references therein).

In spite of these differences, all of these neurotoxins show a large homology in their primary structure: they are weakly basic proteins, with a length of 60-65 residues and four disulfide bridges at similar locations in the sequence. A number of other residues are also conserved or conserve their hydrophobic or hydrophilic character. Secondary-structure predictions indicated that the 3D structures were also likely to be similar (122). This has been confirmed by the X-ray structure of two scorpion neurotoxins belonging to different families: variant-3, isolated from the venom of Centruroides sculpturatus Ewing, a scorpion from the Arizona desert, is only weakly toxic, but may be considered as belonging to the @ family. Its structure was first determined to a resolution of 1.8 A (123, 124) and has recently been refined to 1.2 A, an exceptionallyhigh resolution for a protein structure. The R factor is 19% (121). The a family is represented by its most potent mammal-directed toxin, toxin I1 from Androctonus australis Hector (AaH 11). This crystal structure was refined to R = 18% at 1.8-A resolution (125). The secondarystructure is characterizedin both proteins by a two and a half turn long a-helix (about 10 residues) and a three-stranded antiparallel @-sheet. The helix is roughly parallel to the surface of the @-sheet(Figure 10). Not only do they fold in the same way, but the cores of the two molecules superimpose quite well. Differences are found in the loops, especially of course when the loops are not of the same length due to insertions or deletions of amino acids. There is also a marked difference in the C-terminal region (see Figure 11B,C). In AaH I1 this contributes to the formation of a cavity which is filled with water molecules (125). A 2D proton NMR study of a neurotoxin 66 residues long from a Central Asian scorpion has also yielded a very similar structure (126). Here again, differences were observed mainly in the C-terminal region. Another NMR study was devoted to an anti-insect toxin, AaH IT, from the same venom as the anti-mammal toxin AaH I1 (127). Compared to the anti-mammal toxin, there is an extra tail of five residues at the C-terminal end, which is connected through a disulfide bridge to the second @-strand,while in the anti-mammal toxins a disulfide bridge connects the C-terminal end to the N-terminal part of the molecule. In spite of these differences, the two 3D structures are again very similar, even though their superposition is not quite as good as that of the mammal-directed toxins AaH I1and variant-3. Not surprisingly, the largest differences are once more observed in the C-terminal region, which is thought to be important for the specific interactions with the Na+ channels and the ability to discriminate between these channels in mammals or in insects.

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I

Figure 11. Three scorpion toxins: (A) charybdotoxin, a short-clu toxin; (B)toxin AaH 11; (C) variant-3. Dottec lines indicate 1 ae disulfide bridges, The common structural motif is emphasized by a thick line. There are three conserved disulfic I bridges. Two of them bind a Cye-X-X-X-Cy8motif of the a-helix to a Cya-X-Cy motif of a &strand. A similar pattern was foun in very different proteins or peptides (see text). Reproduced with permission from Bontems et al. (1991) Eur. J.Biochem. 196,194 Copyright 1991 Springer-Verlag.

.

The most interesting characteristicof allthese molecular structures, and probably the most important from a functional point of view, is the distribution of the hydrophobic side chains. Most of them are clustered on one side of the molecule, where they form a rather smooth and conserved-hydrophobic surface with a number of aromatic residues. In AaH 11, four tyrosine rings are aligned in a “herringbone” fashion. Clusters of aromatic residues are also observed in variant-3 and in AaH IT. There is also a number of positively charged side chains, lysines and arginines, some of which have been shown essential for activity: in A& 11,binding to synaptosomes is reduced to less than 0.1% when Lys 58 is chemically modified and to only 1%in the case of Lys 2. These facta are of course strongly reminiscent of the snake venom cardiotoxins. Figure 5 (panels a and c) illustrates the analogy of the side-chain distribution in the two toxin families. There is apparently no sequence homology and no obvious resemblance in the secondary structure (except for the presence of a thee-stranded 8-sheet) nor in the position of the disulfide bridges. But the molecular mass is nearly the same, with the same number of disulfides. Principally, both types are characterized by a similar clustering of hydrophobic residues, and some basic residues play an essential role. Even though the two types of toxins have different targets, a similar mode of interaction with a common membrane component, possibly phospholipids, may be involved. In one proposed model of the interaction the hydrophobic fragment of the scorpion toxin is looked at as the keel of a ship “which allows the incorporation and migration of the neurotoxin molecule into the lipid membrane phase” (119). In the case of cardiotoxins, the f i t loop had been compared to a “hydrophobicanchor”into the phospholipid bilayer (68). It may be significant that the two toxin families have inspired the same kind of marine metaphors! In contrast to cardiotoxins, postsynaptic snake neurotoxins, like erabutoxin,show little resemblance to scorpion

neurotoxins. Presynaptic snake neurotoxins and phospholipases present more topological analogy. The region of the postulated neurotoxicity determinants of phospholipases includes an a-helix and a two-stranded 8-sheet running roughly parallel to the helix (see above), in a way similar to scorpion toxins. However, the helix is longer and follows the 8-sheet in the sequence, while it precedes it in scorpion toxins. Short-ChainScorpion Toxins. More recently, toxins acting on voltage-dependent K+ channels, as do the presynaptic snake toxins, have been isolated from scorpion venoms. These are smaller polypeptides with three disulfide bridges. One representative of this family is noxiustoxin, from the Mexican scorpion Centruroides noxius Hoffmann. It contains 39 amino acids. It is functionally similar to the mamba snake dendrotoxins, since it has been shown that it can displace the binding of dendrotoxin from synaptosomal membranes and, like dendrotoxin, can facilitate acetylcholine release (128). Charybdotoxin is probably the most studied member of this family. This 37-residue-long peptide is known to inhibit high-conductance Ca2+-activated K+ channels present in muscle and neuroendocrinetissues. However, in rat brain, a-dendrotoxin and noxiustoxin block the binding of radiolabeled charybdotoxin, suggesting that the charybdotoxin sites present in brain are more likely associated with inactivating voltage-dependentK+channels (129). On the other hand, the same study showed that iberiotoxin, a selective inhibitor of Ca2+-activated K+ channels, has no effect on charybdotoxin binding on brain, although it is highly homologous (68%)to charybdotoxin (I30). A second class of toxins inhibiting K+ channels is represented by leiurotoxin, ala0 called scyllatoxin, a 31 amino acid long peptide, which blocks a different type of low-conductance Ca2+-activatedK+ channel (131). The binding sites are colocalized with the binding sites of the bee venom neurotoxin apamin.

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Chem. Res. Toxicol., Vol. 6,No.4,1993 399

Chart 11. Sequence Alignment of Long and Short Scorpion Toxins and Insect Defensins (140).

QlTX

spc

EGLPESTPNKSC YS RN

BBBB aaaaaaaaa BBBBB BBBBB The residues conserved in all scorpion toxins are in bold type. The three conserved disulfidebridges and the conserved secondary structure elements are indicated. Var3 = variant-3. ChTX = charybdotoxin. Spc = sapecin. a

Due to this functional diversity, the short scorpion neurotoxins are excellent probes of the various types of K+ channels and can give valuable information on the physiological role of these ion channels. This explains why their 3D structure has received much attention since their discovery. Their relatively small size makes them very suitable for 2D NMR analysis. NMR structures have been published for synthetic (132,133) and natural (134, 135) charybdotoxin, iberiotoxin (1301,and scyllatoxin (136).They all show the same topology, characterized by an a-helix and a two- or three-stranded antiparallel P-sheet (Figure 11A). More subtle structural differences are probably related to the differences in activity. For example, iberiotoxin is less basic than charybdotoxin: one side of the molecule is essentially acid while the other side is basic. The basic side is very similar in the two toxins and is probably the side in contact with the receptor (130). In scyllatoxin, chemical modifications have shown that two arginines of the a-helical region are essential for binding to the Ca2+activated K+channel and for the functional effect of the toxin (131).In contrast, in charybdotoxin, point mutations indicated that the most important residues were again positively charged but situated on the &sheet (137).Thus, binding to different classes of K+ channels involves different sites in these two toxins, in spite of the same general folding (135). A different class of short-chain scorpion toxins is specifically directed toward insects. The structure of the short insectotoxin I& has been determined by 2D NMR (138,139).This too has a 2l/2-turn helix and a 3-stranded &sheet. It differs from the short-chain toxins discussed so far by the presence of a fourth disulfide bridge which connects the beginning of the first strand on the N-terminal side to the a-helix. Even though the polypeptide chain is only 35 residues long, the topology is thus closer to that of the long scorpion toxins. T h e Common Structural Motif in Short and Long Scorpion Toxins. In spite of their different lengths, all the scorpiontoxins described present a very similar folding, characterized by a two- or three-stranded antiparallel @-sheetand an a-helix roughly parallel to the sheet. The relative position of the sheet and the helix is rigidly maintained by two disulfide bonds connecting the helix to the last &strand of the sheet (Figure 11). The two half-cystines in the helix are always separated by three residues and their mates on the @-strandby one residue. Helix and strand run in the same direction. They are separated in the sequence by a stretch of residues forming another 8-strand running in the opposite direction. This antiparallel strand contains the conserved motif Gly-XCys, where the cysteine forms a third conserved disulfide bond with the loop preceding the helix (134).Interestingly, the same characteristics have been found in a completely different type of toxins, namely, insect defensins (140).

These toxins prevent infection by bacteria in insects. Chart 11presents a sequence alignment of short and long scorpion toxins and of an insect defensin. The 2D NMR solution structure of sapecin, a toxin of the defensin family, indeed shows a similar folding (141):this @residuelong polypeptide, with three disulfide bridges, contains an a-helix and a two-stranded 8-sheet in about the same relative location as in the scorpion toxins. Furthermore, there is a hydrophobic surface formed by three alanines and two leucines opposite to a basic residue-rich region. The consensus pattern formed by the sequence portions Cys-X-X-X-Cysand Cys-X-Cys connected by two disulfide bridges has also been found in apamin, the neurotoxin of the honey bee venom. The corresponding three-dimensional motif has been called the "cystine-stabilizeda-helix" (133).In apamin too, the helix is connected to a parallel strand, which however precedes the helix in the sequence (142).The existence of this motif is likelyto be correlated with the ion channel blocking activity, common to apamin and to scorpion toxins. A similar motif was encountered in two small peptides with vasoconstricting activity, namely, human endothelin and snake venom sarafotoxin. However, in these compounds the helix and the strand have opposite directions (133).

Neurotoxins from Other Organisms Cone Snails: Conotoxins. Cone snails living in tropical coral reefs have developed an impressive arsenal of toxins for catching other molluscs, sea worms, and even fish. These toxins are peptides characterized by their small size (12-30 amino acids) and a large content of cysteines (as much as 50%!) forming disulfide bridges. These disulfide bridges ensure the rigidity of the molecule which, due to its small size, would otherwise possess little secondary structure. The initial folding is ensured by a much larger propeptide of which the conotoxin forms the C-terminal part and which is proteolytically cleaved after protein synthesis, the so-called fold-lock-cut pathway (143).Another characteristic of these toxins is their wide diversity and their high specificity. They are directed toward all kinds of ion channels with a high precision, since in many cases they are able to discriminate even between subclasses of channels. Thus, p-conotoxins block voltage-gated sodium channels, but only in skeletal muscles, while the classical Na+ channel toxins tetrodotoxin and saxitoxin bind indiscriminately to muscle and axonal sodium channels. The small size of the toxins, which enables a fast diffusion in the blood of the victim, and their high efficiency are apparently designed for fast immobilization of a rapidly moving prey (143-145). These various characteristics turn the conotoxins into ideal tools for probing the various types of ion channels. Their small size permits easy synthesis and manipulation. There are about 500 species of venomous cone snails, and

400 Chem. Res. Toricol., Vol. 6, No.4,1993

only a few of them have been studied until now. Most attention has been paid to the dangerous fish-hunting Conus geographus. It contains three main classes of toxins: besides the already mentioned pconotoxins, there are the w-conotoxinswhich block the voltage-sensitiveCa2+ channels at vertebrate presynaptic termini, the a-conotoxins which block the acetylcholine receptors at the postsynaptic level, and the conantokins which act on glutamate receptors. 3D structure information is available for a-conotoxins and p-conotoxins. a-Conotoxins are the smallest peptides (13-15 residues). They block the nicotinic acetylcholine receptor in skeletal muscles, the same action as the a-neurotoxins from snake venoms. An a-conotoxin can compete with 1261-labeled a-bungarotoxin for binding sites on muscle preparations (146)and prevent the binding of monoclonal antibodies raised against the a-bungarotoxin binding site of Torpedo cholinoceptors (147).As discussed above (see the section on postsynapic snake neurotoxins), a-conotoxins were found to be precise probes for cross-linking experiments on the nicotinic AChR (54). Several a-conotoxins have been sequenced. A compositional similarity has been found between a-conotoxins and the most conserved central region of erabutoxin (the cysteine-rich region around residue 25) (148). This region is however distinct from the '%oxic site" of erabutoxin. Two independent determinations by 2D NMR of the 3D structure of the same a-conotoxin, conotoxin G1 (or GI), are available (149,150). This peptide has only 13 amino acids, and the structure is constrained by 2 disulfide bridges, so that there is hardly any secondary structure possible, apart from two tight turns centered on residues Pro 5 and Arg 9 (149). Arg 9 has been shown to be important for activity, and it may be one of the two positive charges assumed to be necessary for a tubocurarine-like action on the nicotinic acetylcholine receptor (see above). The other positive charge would be the amino-terminal NH3+ group. The two charges are separated by the aromatic residues His 10 and Tyr 11. In one of the structure determinations the distance between these positive charges is about 15 A, which is larger than in d-tubocurarine [ll 8, (151)1,but the flexibility of the arginine side chain may bring the charges closer together on the receptor (149).In the other NMR determination, the energy minimization refinements resulted in two groups of conformations. One of these groups fulfillsquite well the requirements for a tubocurarine-like interaction with the receptor (150). pConotoxins form a different class of conotoxins, which block voltage-gated sodium channels of skeletal muscles. They consist of 22 amino acids, among which 6 cysteines form 3 disulfide bonds, which again impose strong constraints on the structure. 2D NMR was used in two independent determinations of the solution structure of toxin GIIIA. The two studies agree except that one finds a one-turn helix between residues 14 and 18 (152)where the other shows a less structured coil (153).This portion is linked to the N-terminal part by two disulfide briges 3-15 and 4-20, recalling the "cystine-stabilized a-helix" (see, above, the discussion on scorpion toxins); the overall structure resembles that of apamin from bee venom. The three disulfide bridges form a cage at the center of the molecule. The general shape is that of a disk 6-8, thick and 15 8,in diameter, from which seven Lys and Arg side chains project radially into the solvent. Arg 13 has been shown to be particularly important (154,155).Its guani-

Rees and Bilwes

dinium group may be needed for binding to negative sites of the sodium channels, as do the guanidino groups of the known sodium channel blockers tetrodotoxin and saxitoxin. Arg 13 is part of a flexible loop, which may be able to adapt its conformation upon binding in order to optimize the fit to the receptor (154).An analogous situation has been observed in a sea anemone neurotoxin (see below). Sea Anemones. The venomous apparatus of sea anemones contains proteinaceous toxins, principally membrane-active cytolysins (reviewed in ref 156) and neurotoxins that bind to the voltage-gated sodium channels of excitable tissue. The binding to the sodium channel precludes the channel inactivation and thus prolongs the action potential (see the review in ref 157). The neurotoxins are mostly polypeptides of 45-50 amino acids, crosslinked by 3 disulfide bridges. Smaller peptides (27 amino acids, but still 3 disulfide bridges) have been also found (158).Several toxins of the first kind have been submitted to 2D NMR analysis and their three-dimensional structures refined by molecular dynamics (159-161). One of these toxins has been crystallized, and preliminary X-ray data have been published (162),but to our knowledge, the 3D structure has not been published. Sequence comparisons of the longer polypeptides have shown that they can be divided into two distinct families classified as type 1 and type 2. Calitoxin, a peptide containing 46 amino acids, does not fit into this classification and may represent a third group (163).There is a high sequence homology within each of the two families, but only about 30% homology between them (164).The differences between type 1and type 2 are confirmed by the lack of immunologicalcross-reactivity. In spite of these differences, NMR analyses showed that both types of toxins have the same fold, characterized by a highly twisted 4-stranded antiparallel 8-sheet (Figure 12). From the core of the molecule, secured by the disulfide bridges, several loops stick out, one of which seems to be particularly mobile. This loop includes Arg 14, one of the few amino acids conserved in the two toxin families. This residue seems essential for binding to the receptor and for toxicity, even if this is still open to controversy (157).This recalls very closely the situation in p-conotoxins, which also bind to voltage-gated sodium channels (154). This type of folding with a &sheet, together with the presence of hydrophobic or aromatic patches, is reminiscent of snake venom cardiotoxins. Indeed some, if not all, of these toxins display strong cardiotoxic activity. However, the analogy is even stronger with scorpion toxins: both types of toxins interact with Na+ channels in axons by slowing down the inactivation of the channel. One sea anemone toxin (ATX 11) has been shown to displace the scorpion toxin AaH I1 from its binding site on the synaptosome (165). However, the reverse is not true, even at large concentrations of the scorpion toxin. Even more intriguingly, the number of binding sites of the sea anemone toxin is more than 10 times larger than the number of binding sites of the scorpion toxin. The most likely explanation is that there are at least two types of Na+ channels in synaptosomes, both of them being occupied by the sea anemone toxin, but only one by the scorpion toxin (165). It has been shown recently that the fold of the sea anemone toxins is very similar to a part of the @-barrel structure of serine proteinases (166). Spider Neurotoxins. Spider venoms contain a large variety of synaptic toxins which cause paralysis of the

Invited Reviews

Chem. Res. Toxicol., Vol. 6,No.4, 1993 401

Figure 12. 3D solution structure of two sea anemone toxins (stereoviews): [Panel a (top)] ATX Ia, a toxin of type 1. [Reproduced with permission from Widmer et al. (1989)Proteins 6,357-371(160).Copyright Wiley-Lisa,a division of John Wiley and Sons, Inc.] [Panel b (bottom)] ShI, atoxin of type 2. [Reproducedwith permission from Fogh et al. (1990)J. Biol. Chem. 265,13016-13028 (161). Copyright 1990 The American Society for Biochemistry & Molecular Biology.] In each case, the backbone of the eight best energyrefined structures, with distance restraints from 2D NMR data, is shown. Note the badly defined loop at the top of each molecule. This reflects the lack of long-range NMR restraints for the residues of this loop, probably due to a particularly high flexibility.

insects in which they are injected. As for snakes or other organisms, some of them block postsynaptic receptors, while others affect presynaptic voltage-sensitiveion channels. Examples of the former are the a-agatoxins from the funnel web spider. These are not peptides but arylpolyamines with amolecular mass around 500 Da (167). They block glutamate receptor channels at the neuromuscular junction in insects and provoke reversible paralysis. In the second category are the pagatoxins and the w-agatoxins. pagatoxins cause massive transmitter release at neuromuscular junctions, while w-agatoxins block transmitter release (168,169). p-Agatoxins are made of 36-38 amino acids, among them 8 half-cystines. w-Agatoxins are larger but form a heterogeneous class, ranging from 48 amino acids with 8 cysteines (170) to 76 amino acids with 12 cysteines (169,171). w-Aga IA consists of 66 amino acids and contains 9 cysteines. Despite the odd number of cysteine residues, no cysteine in the reduced form was detected. The explanation came from the primary structure of the precursor, obtained by cDNA cloning. This showed that the mature protein contains a second polypeptidic chain and that the two chains are linked by a disulfide bridge (172). w-Agatoxins have been used as probes of various types of voltage-dependent calcium channels. Their action is distinct from that of

w-conotoxins from snail venoms (1701,but some w-agatoxins have been shown to inhibit the binding of w-conotoxins to presynaptic calcium channels in chick brain synaptosomal membranes (171). Spider venoms may also contain much larger proteins. The most studied is certainly a-latrotoxin from the black widow spider venom (173,174).This neurotoxin causes neurotransmitter release a t the neuromuscular junction or in brain synaptosomes of vertebrates. It is an acidic molecule with a molecular mass of about 130 kDa and no enzymic properties. The primary sequence has been obtained by cloning the cDNA encoding the putative a-latrotoxin precursor (175). This indicated that the polypeptide chain is about 1170residueslong. This protein is known to form channels permeable to monovalent and Ca2+cations in excitable membranes. Its molecular mass is comparable to that of the calcium channels from skeletal muscle membranes, with which it shares a number of properties (174). There is evidence that the flow of calcium ions into the cells caused by the toxin triggers the neurotransmitter secretion. a-Latrotoxin is specific for vertebrates, but a similar insect-specific toxin (a-latroinsectotoxin,M = 120 kDa) has been recently isolated (176). Insects. In addition to PLA2 (177)and the hemolytic toxin melittin (41,the venom of honey bees contains several small neurotoxic peptides. The most toxic to mammals

402 Chem. Res. Toxicol., Vol. 6, No.4,1993

Rees and Bilwes

A5

4

cownz

Figure 13. Schematic representation of apamin,the neurotoxin isolated from the venom of honey bees (142). Note the Cys-XX-X-Cys...Cys-X-Cysmotif, also found in scorpion neurotoxins. Reproduced with permission from LabbB-JulliBet al. (1991)Eur. J.Biochem. 196,639-645(178). Copyright 1991 Springer-Verlag.

is apamin, which has already been mentioned because of its structural analogy with scorpion toxins. It acta as a specific blocker of Ca2+-dependent K+ channels. The binding sites are colocalized with those of the scorpion toxin scyllatoxin (131). A further analogy with scyllatoxin is the existence of two arginines situated on the a-helix, which are essential for the activity (178). The three-dimensional structure of apamin has been determined by NMR (142). Four of the 18 amino acids are cysteines which form 2 disulfide bridges between a short extended strand a t the N-terminal end and a twoturn a-helix on the C-terminal side. The two cysteines of the a-helix are separated by three residues and those of the strand by one residue, as in the scorpion toxins. The two bridges maintain the strand and the helix in an essentially parallel conformation, but they are connected by a 6-residue-long loop instead of an antiparallel strand as in scorpion toxins (Figure 13). The bee venoms contain several other peptides of a size similar to that of apamin (179). Mast cell degranulating peptide (MCD) and tertiapin contain the same consensus pattern Cys-X-Cys and Cys-X-X-X-Cys as apamin. Although they have no neurotoxic properties, spectroscopic data and secondary structure predictions suggested a similar secondary structure (180).

Conclusions

It appears from this review that the information available on three-dimensional structures of animal toxins is still very fragmentary, even if it is developing rapidly. For some animal species, and particularly for snakes, an overview of the different structural types of toxins, incomplete as it may be, has now become possible. For other species, our knowledge is less advanced, but in some cases it has progressed exponentially in the last years, especially for small peptides well suited for NMR studies. The interest in the development of new probes for the various ion channels (the number of newly identified ion channels is growing a t the same rate) is obviously the essential motor of this progression. A good illustration is provided by the cone snail toxins. Animal toxins are very diverse. Nevertheless, they present some common characteristics. Most of them are proteins or peptides, often of small size. Their core is cross-linked and rigidified by disulfide bridges, and one or more flexible loops are frequently observed at the

periphery. The rigid core is in the correct conformation for a good fit to a particular ligand, while the flexible part may allow a fine tuning to optimize the interactions. The general picture emerging is that venomous animals have developed a wide diversity of toxic weapons, directed to virtually every possibletarget in the nervous or muscular systems of their potential preys, with an extraordinary economy of means. The huge number of proteinic toxins contrasts with the relatively small number of molecular folds revealed by the X-ray and NMR structure determinations. The same molecular architecture is used for very different purposes. A good example of this economy is the three-finger fold of many toxins in elapid snake venoms. All the toxins with this folding are about 60amino acids long (70 for the long a-neurotoxins), and their compact core is held together by4 disulfide bridges always in the same position. Yet some of them block the acetylcholine receptor at the neuromuscular junction (neurotoxins), while others bind to some still-mysterious components of excitable membranes (cardiotoxins) or inhibit the enzyme acetylcholinesterase (fasciculins). Toxins acting on muscarinic acetylcholine receptors and calciseptin, which blocks some particular type of calcium channel, are probably of the same structural type. Other important foldings are the phospholipase fold and the trypsin-inhibitor fold. The former is found in phospholipases Az, both mammalian and from snake venoms, in &neurotoxins, and in myotoxins. The trypsininhibitor fold of BPTI is also encountered in mamba dendrotoxins and probably in the small subunit of 0-bungarotoxin. The structure of scorpion toxins has similarly revealed common characteristics, namely an a-helix linked by disulfide bridges to an antiparallel @-sheet. This conformation is also found in completely different toxins, the insect defensins, and the conserved pattern Cys-X-X-XCys in an a-helix, with the cysteines linked to a Cys-XCys group, is present in apamin from bee venom. Sea anemone toxins present another case of unique folding. This has of course important evolutionary implications. It shows that mutations can cause important changes in function, as long as they do not affect the general threedimensional structure. The homology between snake phospholipases or P-neurotoxins and phospholipases Az from mammalian pancreas is consistent with the hypothesis that the venom glands of snakes (as well as the salivary glands of mammals) have evolved from an ancestral gland similar to the pancreas. The fact that phospholipases are found in all snake venoms indicates that they are probably the oldest components of snake venoms. Cardiotoxins of elapid snakes may have resulted from a chain shortening. Postsynaptic neurotoxicity is probably a more recent gain (181). Interesting are also the observed structural similarities between molecules isolated from phylogenetically very distant organisms, like snake toxins and wheat germ agglutinin; or scorpion toxins, bee apamin, and vasoconstricting peptides; or sea anemone toxins and serine proteinases. At least some of these examples may be cases of convergent evolution (166):different organisms have arrived independently at identically folded structures, probably because such structures, due to their stability and their adapatability, provide an optimal support for the function to be exerted.

Acknowledgment. We thank Dr. Derek Logan for his careful and critical reading of the manuscript. We are


Invited Reviews also grateful to Dr. Bengt Westerlund for the color picture shown in Figure 8.

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