Eriksson Mats A L, Roux Benoît
Weill Medical College of Cornell University, Department of Biochemistry, 1300 York Avenue, New York, NY 10021, USA.
Biophys J. 2002 Nov;83(5):2595-609. doi: 10.1016/S0006-3495(02)75270-3.
Computational methods are used to determine the three-dimensional structure of the Agitoxin (AgTx2)-Shaker complex. In a first stage, a large number of models of the complex are generated using high temperature molecular dynamics, accounting for side chain flexibility with distance restraints deduced from thermodynamic analysis of double mutant cycles. Four plausible binding mode candidates are found using this procedure. In a second stage, the quality and validity of the resulting complexes is assessed by examining the stability of the binding modes during molecular dynamics simulations with explicit water molecules and by calculating the binding free energies of mutant proteins using a continuum solvent representation and comparing with experimental data. The docking protocol and the continuum solvent model are validated using the Barstar-Barnase and the lysozyme-antibody D1.2 complexes, for which there are high-resolution structures as well as double mutant data. This combination of computational methods permits the identification of two possible structural models of AgTx2 in complex with the Shaker K+ channel, additional structural analysis providing further evidence in favor of a single model. In this final complex, the toxin is bound to the extracellular entrance of the channel along the pore axis via a combination of hydrophobic, hydrogen bonding, and electrostatic interactions. The magnitude of the buried solvent accessible area corresponding to the protein-protein contact is on the order of 1000 A with roughly similar contributions from each of the four subunits. Some side chains of the toxin adopt different conformation than in the experimental solution structure, indicating the importance of an induced-fit upon the formation of the complex. In particular, the side chain of Lys-27, a residue highly conserved among scorpion toxins, points deep into the pore with its positively charge amino group positioned at the outer binding site for K+. Specific site-directed mutagenesis experiments are suggested to verify and confirm the structure of the toxin-channel complex.
计算方法被用于确定阿吉毒素（AgTx2）与Shaker复合物的三维结构。在第一阶段，利用高温分子动力学生成大量该复合物的模型，通过双突变循环的热力学分析推导的距离约束来考虑侧链灵活性。使用此程序发现了四个合理的结合模式候选物。在第二阶段，通过在含有明确水分子的分子动力学模拟过程中检查结合模式的稳定性，以及使用连续溶剂表示法计算突变蛋白的结合自由能并与实验数据进行比较，来评估所得复合物的质量和有效性。对接协议和连续溶剂模型通过Barstar-Barnase以及溶菌酶-抗体D1.2复合物进行验证，对于这些复合物有高分辨率结构以及双突变数据。这种计算方法的组合允许识别与Shaker钾通道复合的AgTx2的两种可能结构模型，进一步的结构分析提供了支持单一模型的更多证据。在这个最终复合物中，毒素通过疏水、氢键和静电相互作用的组合沿着孔轴与通道的细胞外入口结合。对应于蛋白质-蛋白质接触的埋藏溶剂可及面积的大小约为1000 Å，四个亚基各自的贡献大致相似。毒素的一些侧链采取与实验溶液结构不同的构象，表明复合物形成时诱导契合的重要性。特别是，Lys-27的侧链，这是蝎子毒素中高度保守的一个残基，其带正电荷的氨基深入孔中，位于钾离子的外结合位点。建议进行特定的定点诱变实验来验证和确认毒素-通道复合物的结构。
