Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins

Abstract

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of ␣-cobratoxin (a long-chain ␣-neurotoxin) and heterodimers formed by ␣-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26 -30 in polypeptide loop II of ␣-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the ␣-cobratoxin capacity to compete with ␣-bungarotoxin for binding to Torpedo and ␣7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that ␣-cobratoxin dimer not only interacts with ␣7 nAChR but, in contrast to ␣-cobratoxin monomer, also blocks ␣3␤2 nAChR. In the latter activity it resembles -bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric ␣3␤2 nAChRs. Three-fingered toxins (TFTs)2 are the main components of the Elapidae snake venoms. TFTs consist of one polypeptide chain, their spatial structure being characterized by a hydrophobic core stabilized by four disulfide bridges, which confine three polypeptide loops (fingers). In cobra venom TFTs are represented mainly by ␣-neurotoxins and cytotoxins. So-called short-chain ␣-neurotoxins (60 -62 amino acid residues, 4 intramolecular disulfides) effectively block nicotinic acetylcholine receptors (nAChRs) of muscle-type, and long-chain ␣-neurotoxins (65-75 residues with an additional disulfide in central loop II) block neuronal homopentameric ␣7 nAChR as well (1, 2). These toxins are widely used as tools in the nAChR studies. Cytotoxins, structurally related to short-chain ␣-neurotoxins, manifest another activity; they non-selectively disrupt cell membranes and, thus, kill the cells (3).Another example of TFT interacting with nAChR are -bungarotoxins (-Bgts), minor components of the krait (Elapidae) venom (4, 5). All -Bgts consist of 66 amino acid residues and, similar to long-chain ␣-neurotoxins, contain five disulfide bonds. However, in contrast to ␣-neurotoxins, -Bgts practically do not block muscle-type nAChRs and only weakly act on ␣7 nAChR but with high efficiency interact with ␣3␤2 neuronal receptors (6). Despite the vast array of data on structure-activity relationship for ␣-neurotoxins and -Bgts, it is not yet clear what are the main structural features determining the specificity of a toxin to the particular receptor type. Recently, based on the x-ray structure of ␣-cobratoxin (␣-CT) with acetylcholinebinding protein, Bourne et al. (7) suggested that Lys-29 is the main residue determining the difference in specificity between ␣-neurotoxins and -bungarotoxins. However, A29K mut...

Structural and Functional Characterization of a Novel Homodimeric Three-finger Neurotoxin from the Venom of Ophiophagus hannah (King Cobra)

“…Our laboratory has also reported on a colubrid venom-derived covalently linked heterodimeric 3FTx, irditoxin (13), which was found to target muscle (␣␤␥␦) nAChRs, in sharp contrast to the reported function of elapid dimeric toxins (72). Another distinguishing feature was that the subunits of irditoxin structurally resemble non-conventional 3FTxs (13).…”

“…More recently, other heterodimeric 3FTxs from elapid venoms have been reported, including covalently (disulfide) linked homodimers of a long-chain ␣-neurotoxin (␣-cobratoxin) and heterodimers of ␣-cobratoxin in combination with a variety of three-finger cytotoxins (72). Unlike haditoxin, all of these dimers are formed by covalent bonding (disulfide linkage) of the monomeric units.…”

“…Comparison of Haditoxin with Other Dimeric 3FTxs-Few known examples of dimeric three-finger neurotoxins derived from snake venoms exist (13,37,72). Among them, the most well studied and characterized are the -neurotoxins, known to be composed of two identical monomers held together by noncovalent interactions (77,78).…”

“…Significantly, haditoxin also lacks the fifth disulfide bridge responsible for the cyclization of loop II, which is considered to be a hallmark determinant for the ability of neurotoxins such as ␣-bungarotoxin and -bungarotoxins to interact with their specific neuronal nAChR targets (67,(71)(72)(73)(74). This is somewhat surprising given the high affinity of haditoxin for ␣ 7 -(IC 50 ϭ 0.18 M), ␣ 3 ␤ 2 -(IC 50 ϭ 0.5 M), and ␣ 4 ␤ 2 -(IC 50 ϭ 2.6 M) nAChRs.…”

“…both neuronal ␣ 7 -and ␣ 3 ␤ 2 -nAChRs, unlike monomeric ␣-cobratoxin, which interacts with muscle (␣␤␥␦) and neuronal (␣ 7 ) nAChRs (72).…”