Three-Dimensional Solution Structure of μ-Conotoxin GIIIB, a Specific Blocker of Skeletal Muscle Sodium Channels,

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Abstract
The three-dimensional solution structure of μ-conotoxin GIIIB, a 22-residue polypeptide from the venom of the piscivorous cone snail Conus geographus, has been determined using 2D 1H NMR spectroscopy. GIIIB binds with high affinity and selectivity to skeletal muscle sodium channels and is a valuable tool for characterizing both the structure and function of these channels. Structural restraints consisting of 289 interproton distances inferred from NOEs and 9 backbone and 5 side chain dihedral angle restraints from spin−spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. In addition to the 1H NMR derived information, the 13C resonances of GIIIB were assigned at natural abundance, and hydroxyproline Cβ and Cγ chemical shifts were used to distinguish between the cis and trans peptide bond conformations. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 1.22 Å for the backbone atoms and 2.48 Å for all heavy atoms. For the well-defined region encompassing residues 3−21, the corresponding values were 0.74 and 2.54 Å, respectively. GIIIB adopts a compact structure consisting of a distorted 310-helix, a small β-hairpin, a cis-hydroxyproline, and several turns. The molecule is stabilized by three disulfide bonds, two of which connect the helix and the β-sheet, forming a structural core with similarities to the CSαβ motif [Cornet, B., Bonmatin, J.-M., Hetru, C., Hoffmann, J. A., Ptak, M., & Vovelle, F. (1995) Structure 3, 435−448]. This motif is common to several families of small proteins including scorpion toxins and insect defensins. Other structural features of GIIIB include the presence of eight arginine and lysine side chains that project into the solvent in a radial orientation relative to the core of the molecule. These cationic side chains form potential sites of interaction with anionic sites on sodium channels. The global fold is similar to that reported for μ-conotoxin GIIIA, and the structure of GIIIB determined in this study provides the basis for further understanding of the structure−activity relationships of the μ-conotoxins and for their binding to skeletal muscle sodium channels.