Abstract
The conantokins are a family of small, naturally occurring gamma-carboxyglutamate (Gla)-rich peptides that specifically antagonize the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor. One member of this family, conantokin-G (con-G), undergoes Ca(2+)-mediated self-assembly to form an antiparallel helical dimer. Subunit interactions in this complex are incumbent upon intermolecular Ca(2+) bridging of Gla residues spaced at i, i + 4, i + 7, i + 11 intervals within the monomer. Herein, we further probe the molecular determinants governing such helix-helix interactions. Select variants were synthesized to evaluate the contributions of non-Gla residues to conantokin self-association. Con-G dimerization was shown to be exothermic and accompanied by positive heat capacity changes. Using positional Gla variants of conantokin-R (con-R), a non-dimerizing conantokin, i, i + 4, i + 7, i + 11 Gla spacing alone was shown to be insufficient for self-assembly. The Ca(2+)-dependent antiparallel heterodimerization of con-G and con-T(K7 gamma), two peptides that harbor optimal Gla spacing, was established. Last, the effects of covalently constrained con-G dipeptides on NMDA-evoked current in HEK293 cells expressing combinations of NR1a, NR1b, NR2A, and NR2B subunits of the NMDA receptor were investigated. The antiparallel dipeptide was unique in its ability to potentiate current at NR1a/2A receptors and, like monomeric con-G, was inhibitory at NR1a/2B and NR1b/2B combinations. In contrast, the parallel species was completely inactive at all subunit combinations tested. These results suggest that, under physiological Ca(2+) concentrations, equilibrium levels of con-G dimer most likely exist in an antiparallel orientation and exert effects on NMDA receptor activity that differ from the monomer.
Highlights
The predatory snails of genus Conus are abundant sources of pharmacologically diverse peptides (“conotoxins”) that target numerous neuronal and neuromuscular ion channels
Hundreds of conotoxins have been identified to date, the majority of which are relatively short (10 –30 amino acids) peptides containing 1– 4 intramolecular disulfide bonds that conform to a limited array of bridging frameworks
Conus snails possess the enzymatic machinery to effect numerous post- and co-translational peptide modifications, including C-terminal amidation, epimerization, O-glycosylation, hydroxylation of proline, lysine, and valine, and ␥-carboxylation of glutamate. The latter modification occurs to a high degree in a small family of conotoxins known as the conantokins
Summary
Peptide Synthesis, Purification, and Characterization—The peptides employed in this study, which will be referred to by the numbers designated in Fig. 1, were synthesized as previously described [13], on a model 433A peptide synthesizer (Applied Biosystems, Foster City, CA) at 0.1 mmol scale using PAL resin (Applied Biosystems). All peptides were characterized by analytical HPLC and delayed extraction matrix-assisted laser desorption ionization-time-of-flight mass spectrometry on a Voyager-DE spectrometer (PerSeptive Biosystems, Framingham, MA). The buffer consisted of 10 mM sodium borate, 100 mM NaCl, pH 6.5. Peptide was introduced into the syringe at a concentration of 2.4 mM in buffer consisting of 50 mM Hepes, 150 mM NaCl, and 20 mM CaCl2, pH 7.4. At 3-min intervals, 8 l of this solution was diluted into the sample cell containing 1.4 ml of buffer alone, and the enthalpy changes were recorded. Determination of the Heterodimerization Tendency of 1 and 6— The capacity of 1 and 6 to form heterodimers was determined through previously employed thiol-disulfide oxidation and rearrangement experiments using Cys-containing variants of 1 and 6 [10].
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