alpha-CTx Sequence Source Loops Reference
INDO-PACIFIC FISH HUNTING
INDO-PACIFIC NON-FISH HUNTING ImI GCCSDPRCAWRC* C. imperialis 2 (4/3) McIntosh et al 1994 PnIA GCCSLPPCAANNPDYC* C. pennaceus 2 (4/7) Fainzilber et al 1994 PnIB GCCSLPPCALSNPDYC* C. pennaceus 2 (4/7) Fainzilber et al 1994
EpI GCCSDPRCNMNNPDY^C* C. episcopatus 2 (4/7) Loughnan et al 1998
AuIA GCCSYPPCFATNSDYC* C. aulicus 2 (4/7) Luo et al 1998
AuIB GCCSYPPCFATNPDC* C. aulicus 2 (4/6) Luo et al 1998
AuIC GCCSYPPCFATNSGYC* C. aulicus 2 (4/7) Luo et al 1998
* = Amidated carboxyl terminus; ^ = Sulfated tyrosine, and 'O' in C. ermineus represents trans-4-hydroxyproline.
Click on the conotoxin in the left column above to access the Protein Information Resource (PIR) for further information about that conotoxin. [Courtesy Dr. John Garavelli, PIR, National Biomedical Research Foundation, Washington, DC, USA].
The sequences of 19 known alpha-conotoxins are shown for comparison. Solid lines, confirmed disulfide bridges between conserved cysteine residues (bold type). All of the alpha conotoxins from fish-hunting Indo-Pacific cone snails show high structural homology. Strictly conserved amino acid residues are the disulphides and PA in the middle of the first seven sequences giving a conserved sequence motif XCC(H/N)PACGXX(Y/F)XC*.
In addition to the ten alpha-conotoxins from Indo-Pacific fish-hunting Conus, seven alpha-conotoxins have been purified from non-fish hunting Indo-Pacific species (C. aulicus, C. episcopatus, C. imperialis and C. pennaceus). Three of the latter group, (C. aulicus, C. episcopatus and C. pennaceus) are mollusc eaters while C. imperialis is a worm eater. The alpha-conotoxins from non-fish-hunting Conus species, while having spacing different from that for fish-hunters, retain the same pattern of Cys residues (the "Cys framework"), ie. XnCCXnCXnCXn.
With one exception (alpha-conotoxin SII), the Indo-Pacific alpha conotoxins have two disulphide bonds.
In contrast, the nAChR-targeted conotoxin (PIVA) from the eastern Pacific species C. purpurascens, the purple cone, does not have a Cys framework typical of alpha-conotoxins. Instead, it has three disulphide bonds with the Cys bridging pattern 2-16, 3-11, 14-23. It also has a strikingly different amino acid sequence containing residues of trans-4-hydroxyproline. The so-called alphaA-PIVA not only has a different Cys framework, but the spacing between the first pair of Cys residues and the third Cys is 7 amino acids instead of 3 or 4 amino acids found in the eighteen characterized alpha-conotoxins.
The cysteine residues in all alpha-conotoxins can be aligned. If we represent an alpha-conotoxin schematically as shown above, where loop I consists of the amino acids between the first and third cysteines and loop II consists of the residues between the second and fourth cysteines, then most of the fish-hunting cone venoms have 3 amino acids in loop I and 5 amino acids in loop II. In contrast, alpha EI has 4 amino acids in loop I and 7 amino acids in loop II ( alpha 3/5 and alpha 4/7 groups of alpha conotoxins, respectively). In this respect, alpha-conotoxin EI is similar to alpha-conotoxins PnIA ad PnIB from Conus pennaceus, a snail-hunting species, which also belongs to the alpha 4/7 group. However, although EI and PnI both belong to the 4/7 group, they clearly have different specificity; EI has a phylogenetic specificity much like other alpha conotoxins from fish-hunting cone shells whereas PnIA/B are inactive in vertebrate muscle systems but active in neuronal nicotinic preparations.
alpha Conotoxin MII from the fish-hunting cone, Conus magus, targets the neuronal-type nicotinic receptor rather than the muscle-type nicotinic receptor targeted by the other alpha conotoxins from fish-hunting cones. However, like the other neuronal-nicotinic receptor selective alpha conotoxins, it has 4 amino acids in loop I rather than 3 amino acids.
The neuronal nicotinic receptor targeting
conotoxins (AuIB, EpI, ImI, PnIB and MII) all have 2 disulfide bonds with
4 amino acids in loop I but different numbers of amino acids (3, 6 or 7)
in loop II. This suggests that loop I carries the determinant for neuronal-subtype specificity. In support of this idea, the first loop of neuronal specific conotoxins ImI and EpI contains the sequence SPDR. The neuronal-specific toxin alpha-conotoxin ImI (CTx ImI) has the sequence Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cysteine forms a disulfide bridge to produce a constrained two-loop structure. Quiram and Sine (1998a) found two regions in CTx ImI essential for binding to neuronal alpha7 receptors. The first is the triad Asp-Pro-Arg (DPR) in the first loop, where conservative mutations of each residue diminished affinity by 2-3 orders of magnitude. The second region is the lone Trp in the second loop, where an aromatic side chain is required. The overall results suggest that within the triad of the first loop, Pro positions the flanking Asp and Arg for optimal interaction with one portion of the binding site, while within the second loop, Trp stabilizes the complex through its aromatic ring. Subsequent work (Quiram and Sine 1998b) has identified three pairs of residues in two loops of the neuronal alpha7 acetylcholine receptor that confer selectivity for binding to conotoxin ImI. Two of these pairs, alpha7Trp55/alpha1Arg55 and alpha7Ser59/alpha1Gln59, are within one of the four loops that contribute to the traditional non-alpha subunit face of the muscle receptor binding site. The third pair, alpha7Thr77/alpha1Lys77, is not within previously described loops of either the alpha or non-alpha faces and may represent a new loop or an allosterically coupled loop.
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