Abstract
The three-dimensional solution structure of delta-conotoxin TxVIA, a 27-mer peptide agonist/antagonist of sodium channels, was determined by two-dimensional (1)H NMR spectroscopy with simulated annealing calculations. A total of 20 converged structures of delta-conotoxin TxVIA were obtained on the basis of 360 distance constraints obtained from nuclear Overhauser effect connectivities, 28 torsion angle constraints, and 27 constraints associated with hydrogen bonds and disulfide bonds. The atomic root mean square difference about the averaged coordinate positions is 0.35 +/- 0.07 A for the backbone atoms (N, C(alpha), C) and 0.98 +/- 0.14 A for all heavy atoms of the entire peptide. The molecular structure of delta-conotoxin TxVIA is composed of a short triple-stranded antiparallel beta-sheet. The overall beta-sheet topology is +2x, -1, which is the same as those for other conotoxins. However, the three-dimensional structure of delta-conotoxin TxVIA has an unusual hydrophobic patch on one side of the molecule, which may play an important role in the sodium channel binding. These results provide a molecular basis for understanding the mechanism of sodium channel modulation through the toxin-channel interaction and insight into the discrimination of different ion channels.
Highlights
Voltage-dependent sodium channels are integral plasma membrane proteins responsible for the rapidly rising phase of action potentials in most excitable tissues and are targeted by many neurotoxins
We confirmed that ␦-CTX TxVIA has the same disulfide bonding pattern that is generally observed in other conotoxins, namely, Cys2–Cys17, Cys9–Cys21, and Cys16–Cys26
Structural Comparison of ␦-CTX TxVIA with Other Ion Channel Blockers—In the present study, we have determined the three-dimensional structure of ␦-CTX TxVIA in aqueous solution by using 1H NMR spectroscopy and simulated annealing calculations. ␦-CTX TxVIA is composed of a short triplestranded antiparallel -sheet and several turns
Summary
Peptide Synthesis—␦-CTX TxVIA was chemically synthesized as described previously for TxVII [20], except for a modification of the folding solution. The peptides were concentrated on an ODS column by a medium pressure pump, and ␦-CTX TxVIA was subsequently purified by chromatography on Sephadex G-50F and preparative reversed phase HPLC on an ODS column. The suppression of the solvent resonance was achieved by using the WATERGATE scheme in both the NOESY and TOCSY spectra [25]. For the slowly exchanging backbone amide protons, the sample lyophilized from H2O was redissolved in 2H2O and was identified by analyses of NOESY spectra recorded at time scales of 0.5, 3.0, 6.0, and 12 h. Distance Constraints and Structure Calculations—Interproton distance restraints were obtained from the NOESY spectra with mixing times of either 100 or 300 ms. Pseudo-atoms were used for non-stereospecifically assigned protons, and intra-residue and
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