Conantokin-G and conantokin-T are two paralytic polypeptide toxins originally isolated from the venom of the fish-hunting cone snails of the genus Conus. Conantokin-G and conantokin-T are the only naturally occurring peptidic compounds which possess N-methyl-D-aspartate receptor antagonist activity, produced by a selective non-competitive antagonism of polyamine responses. They are also structurally unusual in that they contain a disproportionately large number of acid labile post-translational ␥-carboxyglutamic acid (Gla) residues. Although no precise structural information has previously been published for these peptides, early spectroscopic measurements have indicated that both conantokin-G and conantokin-T form ␣-helical structures, although there is some debate whether the presence of calcium ions is required for these peptides to adopt this fold. We now report a detailed structural study of synthetic conantokin-G and conantokin-T in a range of solution conditions using CD and 1 H NMR spectroscopy. The three-dimensional structures of conantokin-T and conantokin-G were calculated from 1 H NMR-derived distance and dihedral restraints. Both conantokins were found to contain a mixture of ␣-and 3 10 helix, that give rise to curved and straight helical conformers.

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.