Mangrove catsnake

Three fingers formed by three peptide loops (F1, F2 & F3) [PDB=2H5F]

Denmotoxin is a snake venom protein produced by Boiga dendrophila (mangrove catsnake) which belongs to a family of well studied three-fingered neurotoxins.

Denmotoxin belongs to a family of non-conventional three-finger toxins

Three-finger toxins (3FTXs) are the most common family of snake venom proteins with a conserved structure. The core structure of 3FTXs is formed by joined together by disulphide bridges. Denmotoxin has several features which classify it as a non-conventional 3FTX. It differs structurally from other 3FTX venoms by its seven additional N-terminal amino acid residues; this unusually long N-terminus is unstructured and is hypothesized to gyrate above the core of the protein. There is also an additional fifth disulphide bridge at the first loop of the protein, which is not present in most 3FTXs. Another unique feature of denmotoxin is the twist at the tip of the central loop originating from a kink in a proline residue (Pro40). At the central loop, the charge is also negative, an arginine residue has been replaced with an aspartic acid, which is unusual for the proteins of the family.

Structure

Denmotoxin is a monomeric protein consisting of 77 amino acid residues. Multiple sequence alignment of denmotoxin reveals that the venom belongs to the family of non-conventional 3FTXs. Denmotoxin has 7 additional amino acid residues in its N-terminal when compared to other 3FTXs; the N-terminus is also blocked by a pyroglutamic acid residue. The biological function of the pyroglutamic acid residue is currently unknown.

There are 10 structurally important cysteine-residues in denmotoxin which form five stabilizing . Four of these disulphide bonds are located at the and the fifth at the tip of the first loop. The cysteine residues of all 3FTXs are highly conserved, whereas the other residues within the sequence express high variability. Denmotoxins consists of protruding from the globular core; this structure is typical for 3FTXs. The globular core consists of a triple stranded anti-parallel β-sheet; two of the β-strands in this structure connect to the second loop (central loop) and one β-strand connects to the third loop. There are two highly on the protein: one at the tip of the central loop and one at the 3 first residues of the N-terminus; the expected active site of denmotoxin is at the tip of the central loop.

Denmotoxin shares approximately 30% sequence similarity with other 3FTXs with an exception of exhibiting approximately 50% sequence similarity with another colubrid snake venom α-colubritoxin. Despite the relatively low sequence similarity, denmotoxin possesses all the residues needed to maintain the 3 finger fold. A large part of the sequence similarity between denmotoxin and other 3FTXs is due to the highly conserved disulphides and a number of structurally important residues.

Interaction with nicotinic acetylcholine receptor

Suggested interaction of α-bungarotoxin in binding pocket of nAChR (simplified figure) [PDB=4HQP]

Biochemistry of denmotoxin is unique for its taxon specificity to bird nicotinic acetylcholine receptors (nAChR). Binding of denmotoxin to chick muscle AChR (α1βγδ) is a highly irreversible whereas interaction with identical subunit assembly in mouse AChR is reversible. The reversible binding allows the receptor to function properly, but in the case of irreversible binding nAChR is prevented of natural agonist activation. Previous studies with 3FTXs have shown that the binding of toxin leads to “locking down” of the nACh receptor, preventing required conformational change for ion channel activation and induction of signal.

There are no significant differences in the sequence of functionally important loops A-F of nAChR in mice and chicks. However in the prior region of loop F, chicks have several changes in their amino acid composition leading to introduction of positive charge in the front of the functionally active loop F. This might have important functionality in the attraction and binding of denmotoxin specifically to bird nAChRs.

Denmotoxin lacks the conserved arginine in loop II which is responsible for ACh receptor binding in most of the 3FTX family proteins. Instead this loop carries a mainly due to replacement of arginine to aspartate and presence of an additional glutamate.

Snakes and some other terrestrial animals such as mongoose have achieved resistance to denmotoxin by introducing a mutation to asparagine (e.g. W→N) in one of the aromatic AChR binding residues (see fig.). This allows introduction of N-glycolysation inside the binding pocket leading to spatial inhibition of denmotoxin binding, but still allowing the natural ligand ACh to interact with the receptor.

Conclusions

3FTXs are the most abundant and well characterized snake venom protein family. Whilst the highly conserved three finger structural motif is present in denmotoxin there are minor structural differences in the fold. These changes are responsible for the recognition of target receptors and can modulate both the specificity and toxicity of the venom.