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 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.
Trémeau
O
O
Département d'Ingénierie et d'Etudes des Protéines, DSV, CEA, Saclay, Gif-sur-Yvette, France.
Lemaire
C
C
Drevet
P
P
Pinkasfeld
S
S
Ducancel
F
F
Boulain
J C
JC
Ménez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004870:Erabutoxins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000378:metabolism; D013329:Structure-Activity Relationship; D014101:Torpedo
D009498:Neurotoxins; D011978:Receptors, Nicotinic; C038908:erabutoxin A; D004870:Erabutoxins