Abstract
Conantokin-T (con-T) and conantokin-G (con-G) are peptide toxins derived from Conus venoms, both of which exhibit the N-methyl-D-aspartate (NMDA) antagonist properties. They differ from most conotoxins by their rich γ-carboxyglutamate (Gla) content and lack of cysteine residues. There is a high degree of sequence homology between con-T and con-G, but the solution structures of both peptides are very different in the absence of calcium. CD and 1H NMR studies show that con-G can form a stable helical structure in the presence of divalent metal ions, although the presence of these ions has little effect on the existing helical structure of con-T. 113Cd NMR studies indicate a distinct metal binding behavior for con-G and con-T. There appears to be four Cd resonances in con-G and only one in con-T. It seems that the residue at seventh position play a key role in salt-bridge formation in con-T and calcium network in con-G to stabilize structures. To evaluate the contribution of Lys7 to the helical structure of con-T, we have carried out CD studies of its analog, K7γ-con-T, in which Lys7 was substituted by Gla. It was observed that substitution of Gla residue could increase its ability to bind Ca2+, and induces its helical conformation. Incorporation of this Gla residue seems to introduce more than two additional binding sites on con-T. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Studies of the effects on the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic potential (EPSPNMDA) show that con-T can specifically and irreversibly decrease the EPSPNMDA. The mutated K7γ-con-T also can specifically decrease the EPSPNMDA but in a reversible manner, which is more like con-G.