Hill J M, Alewood P F, Craik D J
Centre for Drug Design and Development, University of Queensland, Brisbane, Australia.
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.
μ-芋螺毒素GIIIB是一种来自食鱼芋螺毒液的由22个氨基酸残基组成的多肽，其三维溶液结构已通过二维¹H NMR光谱法测定。GIIIB与骨骼肌钠通道具有高亲和力和选择性结合，是表征这些通道结构和功能的宝贵工具。由从NOE推断出的289个质子间距离以及来自自旋-自旋耦合常数的9个主链和5个侧链二面角约束组成的结构约束被用作程序X-PLOR中模拟退火计算和能量最小化的输入。除了¹H NMR衍生的信息外，还在天然丰度下对GIIIB的¹³C共振进行了归属，并使用羟脯氨酸Cβ和Cγ化学位移来区分顺式和反式肽键构象。最终的20个结构集对于主链原子在整个分子上的平均成对均方根偏差为1.22 Å，对于所有重原子为2.48 Å。对于包含残基3 - 21的明确区域，相应的值分别为0.74和2.54 Å。GIIIB采用紧凑结构，由一个扭曲的3-10螺旋、一个小的β-发夹、一个顺式羟脯氨酸和几个转角组成。该分子由三个二硫键稳定，其中两个连接螺旋和β-折叠，形成一个与CSαβ基序相似的结构核心[Cornet, B., Bonmatin, J.-M., Hetru, C., Hoffmann, J. A., Ptak, M., & Vovelle, F. (1995) Structure 3, 435 - 448]。这个基序在包括蝎毒素和昆虫防御素在内的几个小蛋白质家族中很常见。GIIIB的其他结构特征包括存在八个精氨酸和赖氨酸侧链，它们相对于分子核心以径向方向伸向溶剂。这些阳离子侧链形成了与钠通道上阴离子位点相互作用的潜在位点。整体折叠与报道的μ-芋螺毒素GIIIA相似，本研究中确定的GIIIB结构为进一步理解μ-芋螺毒素的构效关系及其与骨骼肌钠通道的结合提供了基础。
