Binding of cations to individual gamma-carboxyglutamate residues of conantokin-G and conantokin-T

Conantokin-C (con-G) and conantokin-T (con-T) are naturally occurring gamma-carboxyglutamate (Gla)-containing peptides that interact with multivalent cations in functionally relevant manners. Selective C-13-enrichment of C-gamma and C-delta in each of the Gla residues has allowed metal binding affinities to be measured at individual side chains. Con-T possesses two metal binding sites, one with high affinity at Gla(10)/Gla(14) and another with weak binding at Gla(3)/Gla(4). Con-G contains two sites of comparable low affinity for Ca2+. Analysis of the C-13 line-widths of con-G in the presence of Mg2+ allowed the order of metal binding to be determined, with Gla(10)/Gla(14) loading before the Gla(3)/Gla(4)/Gla(7) cluster. While the variant peptide, ape-con-T[Lys(7)Gla], was shown to have a very low alpha-helical content, this peptide binds a second metal with much greater affinity than wild-type con-T. This provides additional evidence that Gla(7) in con-G is primarily responsible for destabilizing the ape-form, but is an important ligand for metal chelation. The residue-specific alpha-helical stabilities of con-G and con-T in their metal-free and metal-loaded states were estimated by determining rates of proton exchange from backbone peptide bond amides with deuterium atoms from (H2O)-H-2-containing solvents. For both peptides, the lifetimes of protons on several peptide bond amides increased as metals of higher affinity were bound to the peptides, with the longest half-lives found in the region of the alpha-helical turn stabilized by the Gla(10)/Gla(14) metal coordination site. We propose that Gla(10) and Gla(14) constitute the primary tight metal ion binding site in both peptides. This detailed analysis with physiologically relevant metal cations is crucial for deciphering the roles of critical amino acids in the bioactivity of the conantokin peptides.