1. Three-dimensional solution structure of mu-conotoxin GIIIB, a specific blocker of skeletal muscle sodium channels
J M Hill, P F Alewood, D J Craik Biochemistry. 1996 Jul 9;35(27):8824-35. doi: 10.1021/bi960073o.
The three-dimensional solution structure of mu-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 C beta and C gamma 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 A for the backbone atoms and 2.48 A for all heavy atoms. For the well-defined region encompassing residues 3-21, the corresponding values were 0.74 and 2.54 A, respectively. GIIIB adopts a compact structure consisting of a distorted 310-helix, a small beta-hairpin, a cis-hydroxyproline, and several turns. The molecule is stabilized by three disulfide bonds, two of which connect the helix and the beta-sheet, forming a structural core with similarities to the CS alpha beta 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 mu-conotoxin GIIIA, and the structure of GIIIB determined in this study provides the basis for further understanding of the structure-activity relationships of the mu-conotoxins and for their binding to skeletal muscle sodium channels.
2. Roles of basic amino acid residues in the activity of μ-conotoxin GIIIA and GIIIB, peptide blockers of muscle sodium channels
Kazuki Sato, Yoko Yamaguchi, Yukisato Ishida, Yasushi Ohizumi Chem Biol Drug Des. 2015 Apr;85(4):488-93. doi: 10.1111/cbdd.12433. Epub 2014 Sep 30.
To study in detail the roles of basic amino acid residues in the activity of μ-conotoxin GIIIA (μ-GIIIA) and GIIIB (μ-GIIIB), specific blockers of muscle sodium channels, seven analogs of μ-GIIIA, and two analogs of μ-GIIIB were synthesized. μ-GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg1, Arg13, and Arg19) with one, two, and three Lys residues. μ-GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys9, Lys11, Lys16, and Lys19) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg13 of μ-GIIIA was important for the activity, whereas that of Arg19 had little role for biological activity. Although [Arg9,11,16,19]μ-GIIIB showed higher activity than native μ-GIIIB, highly basic [Ala2,12, Arg9,11,16,19]μ-GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.
3. Solution synthesis of mu-conotoxin GIIIB: optimization of the oxidative folding reaction
S Kubo, N Chino, T X Watanabe, T Kimura, S Sakakibara Pept Res. 1993 Mar-Apr;6(2):66-72.
mu-Conotoxin GIIIB, a skeletal muscle sodium channel specific blocker, was synthesized by the solution procedure. The whole molecule, which is composed of 22 amino acid residues including six cysteinyl and three trans-4-hydroxy-L-prolyl residues, was constructed from three segments. After removal of all protecting groups, the hexa-SH peptide was subjected to an oxidative folding reaction at a peptide concentration of 1 x 10(-5) M. Three major products 1, 2 and 3 were formed in a ratio of 1:4:3. Determination of the disulfide structures in each product revealed them to be disulfide isomers, with similar connectivities in the latter two. Study of the biological activities of the three products in mice indicated that only product 1, which is a minor component, has the same potency as the natural product. Analysis of the folding process at a peptide concentration of 1 x 10(-5) M showed that the disulfide bond between Cys10 and Cys15 was initially formed, thus leading to the predominant generation of products 2 and 3. The optimal conditions for the formation of product 1 (mu-conotoxin GIIIB) were obtained by increasing the peptide concentration in the oxidation reaction mixture or by using redox reagents, both of which functioned as promoters for the thiol-disulfide exchange reaction of the mismatched disulfide bond between Cys10 and Cys15.
