Chen Z, Blandl T, Prorok M, Warder S E, Li L, Zhu Y, Pedersen L G, Ni F, Castellino F J
Biomolecular NMR Laboratory and the Montreal Joint Center for Structural Biology, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
J Biol Chem. 1998 Jun 26;273(26):16248-58. doi: 10.1074/jbc.273.26.16248.
The apo- and metal-bound solution conformations of synthetic conantokin-G (con-G, G1Egamma gammaL5Q gamma NQgamma 10LIRgamma K15SN-CONH2, gamma = gamma-carboxyglutamic acid), an antagonist of N-methyl-D-aspartate receptor-derived neuronal ion channels, have been examined by one- and two-dimensional 1H NMR at neutral pH. A complete structure for the Mg2+-loaded peptide was defined by use of distance geometry calculations and was found to exist as an alpha-helix that spans the entire peptide. The alpha-helical nature of Mg2+/con-G was also supported by the small values (<5.5 Hz) of the 3JHNalpha coupling constants measured for amino acid residues 3-5, 8, 9, and 11-16, and the small values (<4 ppb/K) of the temperature coefficients observed for the alphaNH protons of residues 5-17. This conformation contrasted with that obtained for apo-con-G, which was nearly structureless in solution. Docking of Mg2+ into con-G was accomplished by use of the genetic algorithm/molecular dynamics simulation method, employing the NMR-derived Mg2+-loaded structure for initial coordinates in the midpoint calculations. For the 3 Mg2+/con-G model, it was found that binding of one Mg2+ ion is stabilized by oxygen atoms from three gamma-carboxylates of Gla3, Gla4, and Gla7; another Mg2+ is coordinated by two oxygen atoms, one from each of the gamma-carboxylates of Gla7; and a third metal ion through three donor oxygen atoms of gamma-carboxylates from Gla10 and Gla14. As shown from direct metal binding measurements to mutant con-G peptides, these latter two Gla residues probably stabilized the tightest binding Mg2+ ion. Circular dichroism studies of these same peptide variants demonstrated that all Gla residues contribute to the adoption of the Mg2+-dependent alpha-helical conformation in con-G. The data obtained in this investigation provide a molecular basis for the large conformational alteration observed in apo-con-G as a result of divalent cation binding and allow assessment of the roles of individual Gla residues in defining certain of the structure-function properties of con-G.
N-甲基-D-天冬氨酸受体衍生的神经元离子通道拮抗剂——合成芋螺毒素G（芋螺毒素G，G1EγγL5QγNQγ10LIRγK15SN-CONH2，γ=γ-羧基谷氨酸）的脱辅基和金属结合溶液构象，已在中性pH条件下通过一维和二维1H NMR进行了研究。通过距离几何计算确定了负载Mg2+的肽的完整结构，发现其以跨越整个肽的α-螺旋形式存在。对于氨基酸残基3-5、8、9和11-16测得的3JHNα耦合常数较小（<5.5 Hz），以及对于残基5-17的αNH质子观察到的温度系数较小（<4 ppb/K），也支持了Mg2+/芋螺毒素G的α-螺旋性质。这种构象与脱辅基芋螺毒素G的构象形成对比，脱辅基芋螺毒素G在溶液中几乎没有结构。通过使用遗传算法/分子动力学模拟方法，将Mg2+对接至芋螺毒素G中，在中点计算中采用NMR衍生的负载Mg2+的结构作为初始坐标。对于3 Mg2+/芋螺毒素G模型，发现一个Mg2+离子的结合通过来自Gla3、Gla4和Gla7的三个γ-羧酸盐的氧原子得以稳定；另一个Mg2+由两个氧原子配位，一个来自Gla7的每个γ-羧酸盐；第三个金属离子通过来自Gla10和Gla14的γ-羧酸盐的三个供体氧原子配位。如对突变型芋螺毒素G肽的直接金属结合测量所示，后两个Gla残基可能稳定了结合最紧密的Mg2+离子。对这些相同肽变体的圆二色性研究表明，所有Gla残基都有助于芋螺毒素G中依赖Mg2+的α-螺旋构象的形成。本研究中获得的数据为脱辅基芋螺毒素G由于二价阳离子结合而观察到的大的构象改变提供了分子基础，并允许评估各个Gla残基在定义芋螺毒素G的某些结构-功能特性中的作用。
