GENETIC-ENGINEERING OF SNAKE TOXINS - THE FUNCTIONAL SITE OF ERABUTOXIN-A, AS DELINEATED BY SITE-DIRECTED MUTAGENESIS, INCLUDES VARIANT RESIDUES

Using site-directed mutagenesis, we previously identified some residue s that probably belong to the site by which Erabutoxin a (Ea), a sea s nake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) ( Pillet, L., Tremeau, O., Ducancel, F. Drevet, P., Zinn-Justin, S., Pin kasfeld, S., Boulain, J.-C., and Menez, 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 l esser extent at His-6, Ser-9 and Tyr-25 whereas it increases upon muta tion at Ile-36. Other mutations have no effect on Ea affinity. In addi tion, new mutations of the previously explored Ser-8, Asp-81, Arg-33, and Glu-38 better explain the functional role of these residues in Ea. The previous and present mutational analysis suggest that the ''funct ional'' site of Ea covers a homogeneous surface of at least 680 A(2), encompassing the three toxin loops, and includes both conserved and va riant residues. The variable residues might contribute to the selectiv ity of Ea for some AcChoRs, including those from fish, the prey of sea snakes.