Agitoxin is a
toxin
A toxin is a naturally occurring organic poison produced by metabolic activities of living cells or organisms. Toxins occur especially as a protein or conjugated protein. The term toxin was first used by organic chemist Ludwig Brieger (1849– ...
found in the
venom
Venom or zootoxin is a type of toxin produced by an animal that is actively delivered through a wound by means of a bite, sting, or similar action. The toxin is delivered through a specially evolved ''venom apparatus'', such as fangs or a sti ...
of the scorpion ''
Leiurus quinquestriatus hebraeus'' (yellow scorpion). Other
toxins
A toxin is a naturally occurring organic poison produced by metabolic activities of living cells or organisms. Toxins occur especially as a protein or conjugated protein. The term toxin was first used by organic chemist Ludwig Brieger (1849–1 ...
found in this species include
charybdotoxin
Charybdotoxin (CTX) is a 37 amino acid neurotoxin from the venom of the scorpion '' Leiurus quinquestriatus hebraeus'' (''deathstalker'') that blocks calcium-activated potassium channels. This blockade causes hyperexcitability of the nervous syst ...
(CTX). CTX is a close homologue of Agitoxin.
Structure
Agitoxin can be purified using
HPLC
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pa ...
techniques.
Primary structure:
Three types of agitoxin can be distinguished, each identified as comprising 38
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s. They are highly homologous, differing only in the identity of the residues at positions 7, 15, 29 and 31.
![Agitoxinalignment](https://upload.wikimedia.org/wikipedia/commons/4/4b/Agitoxinalignment.png)
*Agitoxin-1 Gly-Val-Pro-Ile-Asn-Val-Lys-Cys-Thr-Gly-Ser-Pro-Gln-Cys-Leu-Lys-Pro-Cys-Lys-Asp-Ala-Gly-Met-Arg-Phe-Gly-Lys-Cys-Ile-Asn-Gly-Lys-Cys-His-Cys-Thr-Pro-Lys (GVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHCTPK,
molecular weight
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...
= 4014.87
Da, molecular formula = C
169H
278N
52O
47S
7)
*Agitoxin-2 Gly-Val-Pro-Ile-Asn-Val-Ser-Cys-Thr-Gly-Ser-Pro-Gln-Cys-Ile-Lys-Pro-Cys-Lys-Asp-Ala-Gly-Met-Arg-Phe-Gly-Lys-Cys-Met-Asn-Arg-Lys-Cys-His-Cys-Thr-Pro-Lys (GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK, molecular weight = 4090.95 Da, molecular formula = C
169H
278N
54O
48S
8)
*Agitoxin-3 Gly-Val-Pro-Ile-Asn-Val-Pro-Cys-Thr-Gly-Ser-Pro-Gln-Cys-Ile-Lys-Pro-Cys-Lys-Asp-Ala-Gly-Met-Arg-Phe-Gly-Lys-Cys-Met-Asn-Arg-Lys-Cys-His-Cys-Thr-Pro-Lys (GVPINVPCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK, molecular weight = 4100.98 Da, molecular formula = C
171H
280N
54O
47S
8,
CAS Number
A CAS Registry Number (also referred to as CAS RN or informally CAS Number) is a unique identification number assigned by the Chemical Abstracts Service (CAS), US to every chemical substance described in the open scientific literature. It inclu ...
155646-23-4)
Secondary and tertiary structure:
Agitoxin consists of a triple-stranded antiparallel
beta-sheet
The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a g ...
in which the C-terminal strand sits in the centre of the sheet, and a single
alpha-helix
The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
covering one face of the beta-sheet (see image on the right). The
cysteine side chains connect the beta-sheet and the helix via
disulphide bonds
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
to form the core of the molecule.
The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins, classified as 'Scorpion short toxins' by
Pfam
Pfam is a database of protein families that includes their annotations and multiple sequence alignments generated using hidden Markov models. The most recent version, Pfam 35.0, was released in November 2021 and contains 19,632 families.
Uses
...
.
This fold, and the location of the disulphide bonds, are a shared element between toxins stemming from arthropods. The structure of agitoxin-2, has been determined by NMR.
Function
Agitoxin binds to the
Shaker ''K''
+ channel in ''
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
'' as well as to its mammalian homologue. It blocks this channel by binding with high affinity (''K''
d < 1
nmol/
L) to its external vestibule.
This high affinity to the ‘Shaker K’ channel is dependent on the residues of Arg
24, Lys
27, and Arg
31.
The ability of the agitoxin to block the ‘Shaker K’ channel suggests a docking mechanism whereby the toxin sits on the channel and then prevents its opening through flexible movements of the side chains thereby allowing for various protein-protein complexes to be enacted.
AgTx2 has been found to undergo conformational changes when bound to the ‘Shaker K’ channel which suggests the
induced fit
Enzyme catalysis is the increase in the rate of a process by a biological molecule, an "enzyme". Most enzymes are proteins, and most such processes are chemical reactions. Within the enzyme, generally catalysis occurs at a localized site, call ...
model may be present in toxin-channel interaction. This mode of interaction goes along with and explains the flexible side chains.
The
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
make up of the toxin determines the way each interacts with
Shaker K channels.
In Agitoxin, for example, the arginine interacts electrostatically with the aspartate of the ‘Shaker K’ channel.
The final blocking of the pore in the Shaker K + channel occurs via the side chain of Lys
27.
Lys
27 interacts with the Shaker K by plugging the selectivity filter and hydrogen bonding with carbonyls of Tyr
395 of each potassium channel subunit.
Important stabilizing hydrogen bonding between various residues on the AgTx2 and the subunits of the channel hold the toxin-channel complex together. Mutations in Arg24Ala, Lys27Met, and Asn30Ala increased the dissociation, or break-down, of the complex, suggesting they all play important roles in holding the toxin on the channel.
References
Further reading
*
*
*
{{Toxins
Neurotoxins
Ion channel toxins
Scorpion toxins