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

Abstract

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (␣␤␥␦) and neuronal (␣ 7 , ␣ 3 ␤ 2 , and ␣ 4 ␤ 2 ) nicotinic acetylcholine receptors (nAChRs) with highest affinity for ␣ 7 -nAChRs. The high resolution (1.5 Å ) crystal structure revealed haditoxin to be a homodimer, like -neurotoxins, which target neuronal ␣ 3 ␤ 2 -and ␣ 4 ␤ 2 -nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic shortchain ␣-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain ␣-neurotoxins and -neurotoxins notwithstanding, haditoxin exhibited unique blockade of ␣ 7 -nAChRs (IC 50 180 nM), which is recognized by neither shortchain ␣-neurotoxins nor -neurotoxins. This is the first report of a dimeric short-chain ␣-neurotoxin interacting with neuronal ␣ 7 -nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.Snake venoms are a rich source of pharmacologically active proteins and polypeptides targeting a variety of receptors with high affinity and specificity (1). Because of their high specificity, some of these molecules have contributed significantly (a) to the isolation and characterization of different receptors and their subtypes in the field of molecular pharmacology and (b) as lead compounds in the development of therapeutic agents (2, 3). For example, the discovery of ␣-bungarotoxin, a postsynaptic neurotoxin from the venom of Bungarus multicinctus, led to the identification of the nicotinic acetylcholine receptor (nAChR), 3 the first isolated receptor protein (4) as well as the first one to be characterized electrophysiologically (5) and biochemically (6, 7). Subsequently, it was also used to characterize several other nAChRs (8 -10).Snake venom proteins can be broadly classified as enzymatic and non-enzymatic proteins. Three-finger toxins (3FTxs) are the largest group of non-enzymatic snake venom proteins (1, 11). They are most commonly found in the venoms of elapid and hydrophiid snakes. Recently, our laboratory has also demonstrated the presence of 3FTxs from colubrid venoms (12, 13), and 3FTx transcripts have been found in the venom gland transcriptome of viperid snakes (14, 15). The proteins in this family of toxins share a common structural scaffold of three ␤-sheeted loops emerging from a central core (11,16). Despite the overall similarity in structure, these proteins have diverse functional properties. Members of this family include neurotoxins targeting the cholinergic system (7, 11, 16), cytotoxins/cardiotoxins interacting with the cell membranes (17), calciseptine and related toxins that block th...

Dimeric α-Cobratoxin X-ray Structure

“…The heteromeric dimer of ␣-cobratoxin and cytotoxin has an activity similar to that of ␣CT-␣CT against the ␣7 nAChR and is more active against ␣3␤2 nAChRs. Our results demonstrate that at least one Cys 26 -Cys 30 disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by ␣7 nAChR, although it is less important for interaction of covalent TFT dimers with the ␣3␤2 nAChR.…”

“…Here, we report the ␣CT-␣CT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys 3 in one protomer and Cys 20 of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys 26 and Cys 30 in the central loops II, have the same positions as in monomeric ␣-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the ␣CT-␣CT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the -bungarotoxin solution structure.…”

“…The three-finger fold is essentially preserved in each protomer, but the arrangement of the ␣CT-␣CT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the -bungarotoxin solution structure. Selective reduction of Cys 26 -Cys 30 in one protomer does not affect the activity against the ␣7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents ␣7 nAChR recognition. On the contrary, reduction of one or both Cys 26 -Cys 30 disulfides in ␣CT-␣CT considerably potentiates inhibition of the ␣3␤2 nAChR by the toxin.…”

“…Selective reduction of Cys 26 -Cys 30 in one protomer does not affect the activity against the ␣7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents ␣7 nAChR recognition. On the contrary, reduction of one or both Cys 26 -Cys 30 disulfides in ␣CT-␣CT considerably potentiates inhibition of the ␣3␤2 nAChR by the toxin. The heteromeric dimer of ␣-cobratoxin and cytotoxin has an activity similar to that of ␣CT-␣CT against the ␣7 nAChR and is more active against ␣3␤2 nAChRs.…”

Snake Bite in Jersey Cattle; a Case Report

“…Venom of Indian Cobra predominantly contains neurotoxins, but it also contains cardiotoxin (Behler, 1989). Toxic constituents are mainly proteins and polypeptides (Roy et al, 2010). This clinical article reports a case of cobra envenomation in a female jersey cow.…”