A single amino acid replacement results in the Ca2+-induced self-assembly of a helical conantokin-based peptide
A single amino acid replacement results in the Ca2+-induced self-assembly of a helical conantokin-based peptide
复制标题
DOI：
10.1021/bi048796s
复制
发表时间：
2004-10-19
期刊：
影响因子：
2.9
通讯作者：
Prorok, M
中科院分区：
文献类型：
--
作者：
Dai, QY;Castellino, FJ;Prorok, M
圆锥螺蛋白是圆锥螺属海洋蜗牛毒液中富含 γ-羧基谷氨酸 (Gla) 的短肽成分（17-27 个氨基酸残基）。它们表现出高 apo 和/或 Ca2+ 诱导的螺旋性，并可作为 N-甲基-D-天冬氨酸受体 (NMDAR) 的有效和选择性抑制剂。我们之前已经确定，锥囊蛋白之一，锥囊蛋白-G (con-G)，在存在 Ca2+ 的情况下自缔合，具有高特异性的反平行链方向 [Dai, Q., Prorok, M., 和 Castellino, F. J. (2004) J. Mol.生物。 336、731-744]。二聚化似乎是由以下残基配对之间的螺旋间 Ca2+ 配位驱动的：Gla(3)-Gla(14)、Gla(7)-Gla(10')、Gla(10)-Gla(7') 和 Gla(14)-Gla(3')。 Conantokin 家族的第二个成员，conantokin-T (con-T)，在 21 个氨基酸中的 8 个与 con-G 具有序列同一性，包括 4 个 Gla 残基。尽管有这些相似之处，con-T 和 con-G 之间仍存在一些主要和次要结构差异。特别值得注意的是，con-T 在第 7 位包含 Lys，而不是 Gla。此外，与 con-G 不同，con-T 不会发生 Ca(2+) 触发的自组装。在本研究中，采用沉降平衡超速离心来证明 con-T 的单一氨基酸替代类似物 con-T[K7gamma] 在 Ca2+ 存在下、pH 值与 Gla 羧酸盐基团的电离一致时呈现二聚超结构。此外，HPLC 监测的硫醇二硫化物折叠和重排分析表明，非共价复合物中的相对链排列偏好是反平行的。我们的结果表明，con-T[K7gamma] 中的链间 Ca2+ 配位依赖于 Gla 残基的“i，i + 4，i + 7，i + 11”排列，如天然 con-G 中所发生的那样。
Conantokins are short (17-27 amino acid residues), gamma-carboxyglutamate (Gla)-rich peptide components of the venoms of marine snails of the genus Conus. They display high apo and/or Ca2+-induced helicity and act as potent and selective inhibitors of the N-methyl-D-aspartate receptor (NMDAR). We have previously established that one of the conantokins, conantokin-G (con-G), self-associates in the presence of Ca2+ with high specificity for antiparallel chain orientation [Dai, Q., Prorok, M., and Castellino, F. J. (2004) J. Mol. Biol. 336, 731-744]. The dimerization appears to be driven by interhelical Ca2+ coordination between the following residue pairings: Gla(3)-Gla(14), Gla(7)-Gla(10'), Gla(10)-Gla(7'), and Gla(14)-Gla(3'). A second member of the conantokin family, conantokin-T (con-T), shares sequence identity with con-G at 8 of 21 amino acids, including 4 Gla residues. These similarities notwithstanding, several primary and secondary structural differences exist between con-T and con-G. Particularly notable is that con-T contains a Lys, rather than a Gla, at position 7. Moreover, unlike con-G, con-T does not undergo Ca(2+)triggered self-assembly. In the present study, sedimentation equilibrium ultracentrifugation is employed to demonstrate that a single amino acid replacement analogue of con-T, con-T[K7gamma], assumes a dimeric superstructure in the presence of Ca2+ at pH values consistent with the ionization of Gla carboxylate groups. Further-more, HPLC-monitored thiol-disulfide folding and rearrangement assays with Cys-containing con-T variants suggest that the relative chain alignment preference in the noncovalent complex is antiparallel. Our results suggest that interchain Ca2+ coordination in con-T[K7gamma] is incumbent upon an "i, i + 4, i + 7, i + 11" arrangement of Gla residues, as occurs in native con-G.