Han K H, Hwang K J, Kim S M, Kim S K, Gray W R, Olivera B M, Rivier J, Shon K J
Biomolecular Structure Research Unit, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea.
Biochemistry. 1997 Feb 18;36(7):1669-77. doi: 10.1021/bi962301k.
A high-resolution solution conformation of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA, GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been determined by two-dimensional 1H NMR methods and distance geometry calculations. The total of 324 NOE-derived interproton distance restraints including 33 long-range NOE restraints as well as 11 phi and 7 chi 1 torsion angle restraints was used for computation of structures. Back-calculation from the experimental NOE spectrum has provided 49 new NOE restraints and yielded the final R-factors of Ra = 0.641 and Rb = 0.157. The final RMSD values are 0.90 and 1.16 A for the backbone and the heavy atoms, respectively. The C-terminal half of the molecule involving the residues 12-24 is extremely well-defined with a backbone RMSD value of 0.56 A, whereas the N-terminal 3-11 disulfide loop is relatively flexible, possessing a backbone RMSD value of 1.09 A. The [Pro 7,13] alpha A-conotoxin PIVA does not contain any significant secondary structure although the 21S-24G nearly completes one turn of a 3(10) helix. The overall protein fold is largely maintained by the three disulfide bridges of 2-16, 3-11, and 14-23. The presence of the three disulfide bridges imposes geometric constraints that force the molecule to form six continuous bends involving the following residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q. The overall shape of the [Pro 7,13] alpha A-conotoxin PIVA can be described as an "iron". Residues 15S-19R form a loop that protrudes out of the "bottom plate" formed by the rest of the protein and constitute the handle of the iron. The N-terminal tip of the molecule is relatively immobile due to attractive electrostatic interactions between the gamma-hydroxyl group of 20 Hyp and the phenolic hydroxyl group of 22Y. The flexible 3-11 disulfide loop consists mostly of hydrophobic residues, while the best-defined 14-23 disulfide loop contains the highly charged hydrophilic 15S-19R "handle" domain exposed to the exterior of the protein. Binding to nicotinic acetylcholine receptor can be mediated through two different types of interactions: one involving the aromatic hydrophobic residues such as 6Y and 12H and the other involving the positively charged hydrophilic side chain of the 19R. The side chain of the 19R in the [Pro 7, 13] alpha A-conotoxin PIVA and that of the 9R of the alpha-conotoxin G1, and also the side chains of the 12H and 6Y in the former and those of 10H and 11Y in the latter can be aligned to point to the same direction when the corresponding backbone atoms are superimposed to an RMSD value of 2.5 A.
通过二维¹H NMR方法和距离几何计算，已确定了一种新型芋螺毒素[Pro 7,13]αA-芋螺毒素PIVA（GCCGSYPNAACHPCSCKDROSYCGQ-NH₂）的高分辨率溶液构象。总共324个源自核Overhauser效应（NOE）的质子间距离约束，包括33个长程NOE约束以及11个φ和7个χ₁扭转角约束，用于结构计算。根据实验性NOE谱进行的反向计算提供了49个新的NOE约束，并得出最终的R因子：Ra = 0.641和Rb = 0.157。主链和重原子的最终均方根偏差（RMSD）值分别为0.90 Å和1.16 Å。分子的C端一半（涉及残基12 - 24）定义极为明确，主链RMSD值为0.56 Å，而N端3 - 11二硫键环相对灵活，主链RMSD值为1.09 Å。[Pro 7,13]αA-芋螺毒素PIVA不包含任何显著的二级结构，尽管21S - 24G几乎完成了一个3(10)螺旋的一圈。整体蛋白质折叠主要由2 - 16、3 - 11和14 - 23的三个二硫键维持。这三个二硫键的存在施加了几何约束，迫使分子形成六个连续的弯曲，涉及以下残基：3C - 5S、7P - 10A、12H - 14C、15S - 17K、17K - 19R和21S - 25Q。[Pro 7,13]αA-芋螺毒素PIVA的整体形状可描述为一个“熨斗”。残基15S - 19R形成一个环，从由蛋白质其余部分形成的“底板”中突出，构成熨斗的把手。由于20羟脯氨酸的γ-羟基与22Y的酚羟基之间存在有吸引力的静电相互作用，分子的N端相对固定。灵活的3 - 11二硫键环主要由疏水残基组成，而定义最明确的14 - 23二硫键环包含暴露于蛋白质外部的高度带电的亲水性15S - 19R“把手”结构域。与烟碱型乙酰胆碱受体的结合可通过两种不同类型的相互作用介导：一种涉及芳香族疏水残基，如6Y和12H，另一种涉及19R的带正电荷的亲水性侧链。当相应的主链原子叠加至RMSD值为2.5 Å时，[Pro 7,13]αA-芋螺毒素PIVA中19R的侧链与α-芋螺毒素G1中9R的侧链，以及前者中12H和6Y的侧链与后者中10H和11Y的侧链可排列成指向同一方向。
