Using site-directed mutagenesis, we previously identified some residues that probably belong to the site by which Erabutoxin a (Ea), a sea snake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) (Pillet, L., Trémeau, O., Ducancel, F. Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Ménez, A. (1993) J. Biol. Chem. 268, 909-916). We have now studied the effect of mutating 26 new positions on the affinity of Ea for AcChoR. The mutations are F4A, N5V, H6A, Q7L, S9G, Q10A, P11N, Q12A, T13V, T14A, K15A, T16A, delta S18, E21A, Y25F, Q28A, S30A, T35A, I36R, P44V, T45A, V46A, K47A, P48Q, I50Q, and S53A. Binding affinity decreases upon mutation at Gln-7, Gln-10 and to a lesser extent at His-6, Ser-9 and Tyr-25 whereas it increases upon mutation at Ile-36. Other mutations have no effect on Ea affinity. In addition, new mutations of the previously explored Ser-8, Asp-31, Arg-33, and Glu-38 better explain the functional role of these residues in Ea. The previous and present mutational analysis suggest that the "functional" site of Ea covers a homogeneous surface of at least 680 A2, encompassing the three toxin loops, and includes both conserved and variant residues. The variable residues might contribute to the selectivity of Ea for some AcChoRs, including those from fish, the prey of sea snakes.

We identified the residues that are important for the binding of ␣-dendrotoxin (␣DTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys 5 and Leu 9 decreased affinity more than 1000-fold, and substitutions of Arg 3 , Arg 4 , Leu 6 , and Ile 8 by 5-30-fold. Substitution of Lys 5 by norleucine or ornithine also greatly altered the binding properties of ␣DTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type ␣DTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser 38 and Arg 46 also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Venomous animals from four distinct phyla produce small toxic proteins that block a variety of Kv1 voltage-gated potassium channels. These are the scorpions (1), sea anemones (2-4), marine cone snails (5), and snakes (6 -8), which are arthropods, cnidarians, molluscs, and chordates, respectively. At least four different folds, the sizes of which range from approximately 30 to 60 residues, are associated with these different potassium channel-blocking toxins. These are (i) the ␣/␤-toxin fold, which is found in scorpion toxins, such as charybdotoxin (9); (ii) the fold that comprises two short helices and is only adopted by toxins from sea anemone toxins such as ShK (10) and BgK (11); (iii) the -conotoxin fold, which has three ␤-sheet strands and is adopted by -conotoxin from cone snails (12, 13); and (iv) the BPTI 1 -type fold (14), composed of two short helices and a two-stranded ␤-sheet, which is adopted by the snake dendrotoxins (15-17) and probably by the sea anemone kalicludines (4).Although structurally unrelated, the Kv1 channel-blocking toxins produced by scorpions, snakes, sea anemones, and snails all are likely to bind to the peptide loop between the membranespanning segments S5 and S6 of Kv1 channels (18 -25, 11). Therefore, all of these toxins may possess a functional surface that is complementary to this loop, an observation that raises the question as to how similar these surfaces are from one toxin to another. The answer to such a question may not only shed light on the evolution of these toxins but should also help characterize the surface by which Kv1 channels interact with these toxins. Mutational analyses have finely delineated the functional sites of scorpion toxins (26) and sea anemone toxins (11,25). Although the sea anemone and scorpion toxins are not structurally related, their functional sites share some similarities. They are all flat surfaces of comparable size (ϳ700 Å 2 ) with five functionally important residues, including a similar critical function...

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We investigated the interaction of toxin gamma, a cardiotoxin from the venom of the elapid Naja nigricollis, with perdeuterated dodecylphosphocholine (DodPCho) micelles using standard two-dimensional proton NMR spectroscopy. The proton spectrum resonances of the micelle-bound toxin gamma were assigned, and the chemical shifts of the backbone and side-chain protons were compared with those determined in the absence of DodPCho. We observed that DodPCho induced large chemical shift changes on residues localized on the hydrophobic face of the toxin. These changes are not associated with conformational changes of the toxin. However, the micellar environment may induce some stabilization of the triple-stranded beta sheet, the major component of the protein structural core. Since the proton NMR spectrum of toxin alpha, a structurally related neurotoxin extracted from the same venom, was unaffected by the presence of the micelles, we came to the conclusion that the observed effects are specific to cardiotoxins. The present results give direct evidence of the contribution of the hydrophobic face of the toxin to the toxic site and further suggest a possible mechanism of action of cardiotoxin on biological bilayers.