Abstract
The solution structure of the Ca2+-loaded conantokin-T (con-T), a gamma-carboxyglutamate (Gla)-containing 21-residue peptide (NH2-G1EgammagammaY5QKMLgamma10NLRgammaA15EVKKN20A-CONH2,gam ma = Gla), has been elucidated by use of distance geometry calculations with experimental distances derived from two-dimensional 1H NMR spectroscopy. An end-to-end alpha-helix was the dominant conformation in solution, similar to that of apo-con-T, except that reorientation of several side chains occurred in the Ca2+-coordinated complex. The most notable examples of this were those of Gla10 and Gla14, which were more optimally positioned for complexation with Ca2+. In addition to the stabilization offered to the alpha-helix by Ca2+ binding, hydrophobic clustering of the side chains of Tyr5, Met8, Leu9, and Leu12, and ionic interactions between Lys7 and Gla3/Gla10 and between Arg13 and Gla14, along with hydrogen bonding between Gln6 and Gla10, were among the side chain interactions likely playing a significant role in maintenance of the alpha-helical conformation. Docking of Ca2+ in the con-T structure was accomplished using genetic algorithm-molecular dynamics simulation approaches. The results showed that one Ca2+ ion is most likely coordinated by four side chain oxygen atoms, two each from Gla10 and Gla14. Another bound Ca2+ ion has as its donor sites three oxygen atoms, two from Gla3 and one from Gln6. To examine the functional roles of the individual Gla residues, a series of variant peptides have been synthesized with Ala substituted for each Gla residue, and several properties of the resulting variants have been examined. The data obtained demonstrated the importance of Gla10 and Gla14 in stabilizing binding of the highest affinity Ca2+ site and in governing the conformational change induced by Ca2+. The critical nature of Gla3 and Gla4 in inhibition of the spermine-induced potentiation of the binding of MK-801 to open ion channels of the N-methyl-D-aspartate receptor was established, as well as the role of Gla4 in stabilizing the apo-con-T alpha-helical conformation.
Highlights
The solution structure of the Ca2؉-loaded conantokin-T, a ␥-carboxyglutamate (Gla)-containing 21-residue peptide (NH2-G1E␥␥Y5QKML␥10NLR␥A15EVKKN20ACONH2,␥ ؍Gla), has been elucidated by use of distance geometry calculations with experimental distances derived from two-dimensional 1H NMR spectroscopy
In addition to the stabilization offered to the ␣-helix by Ca2؉ binding, hydrophobic clustering of the side chains of Tyr5, Met8, Leu9, and Leu12, and ionic interactions between Lys7 and Gla3/ Gla10 and between Arg13 and Gla14, along with hydrogen bonding between Gln6 and Gla10, were among the side chain interactions likely playing a significant role in maintenance of the ␣-helical conformation
Con-T, it interacts with these cations, does not undergo as dramatic a conformational change, because apo-con-T is already highly organized in an ␣-helical conformation [12, 14]
Summary
Vol 273, No 13, Issue of March 27, pp. 7512–7522, 1998 Printed in U.S.A. The Roles of Individual ␥-Carboxyglutamate Residues in the Solution Structure and Cation-dependent Properties of Conantokin-T*. A general class of Conus peptides, the conotoxins, which contain a relatively high number of disulfide bonds, interact with nicotinic acetylcholine receptors (␣-conotoxins), voltage-sensitive Naϩ channels (conotoxins), and voltage-sensitive Ca2ϩ channels (-conotoxins) [6] Conantokins, such as the 17-amino acid residue peptide con-G [7] and the homologous 21-residue con-T [1], are Gla-containing peptides without disulfide bonds. In the present phase of our efforts in this area, we have defined the threedimensional conformation of the Ca2ϩ1⁄7con-T complex and have employed mutant con-T peptides to attempt to understand the role of individual Gla residues in defining its metal binding properties and its bioactivity.
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