Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNA synthetase
Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNA synthetase
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DOI：
10.1006/jmbi.2000.4285
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发表时间：
2001-02-16
影响因子：
5.6
通讯作者：
Karplus, M
中科院分区：
文献类型：
--
作者：
Archontis, G;Simonson, T;Karplus, M
氨基酰-tRNA合成酶(AARs)与特定氨基酸的结合是正确翻译遗传密码所必需的。已提出在氨基酰-tRNA合成酶中设计一种修饰的专一性，作为一种将人工氨基酸残基结合到葡萄蛋白质中的方法。在前文中，用分子动力学自由能模拟比较了底物天冬氨酸和类似物天冬氨酸与天冬氨酸氨基-tRNA合成酶的结合。分子动力学与泊松-玻尔兹曼自由能计算相结合代表了一种成本较低的方法，适合于在工程工作中检查多个活性位点突变。在这里，Poisson-Boltzmann计算天冬氨酸-tRNA合成酶的自由能首先通过它们再现选定的分子动力学结合自由能差异的能力来验证，然后用来研究ASN与天然和突变的天冬氨酸-tRNA合成酶结合的可能性。泊松-玻尔兹曼自由能的分量分析被用来确定决定结合亲和力的特定相互作用。结合使用分子动力学自由能模拟来深入研究一个结合过程，然后结合分子动力学和泊松-玻尔兹曼自由能计算来研究一系列相关的配体或突变，被建议作为蛋白质或配体设计的范例。分析了在同源天冬酰胺合成酶中观察到的交替的头到尾的方向的天冬氨酸结合，发现比先前研究的类似天冬氨酸的方向更稳定。新的取向可能不适合催化。识别天冬氨酸侧链的保守活性位点赖氨酸(在大肠杆菌中为Lys198)被改变为亮氨酸残基，位于天冬酰胺-tRNA合成酶的相应位置。有趣的是，天冬氨酸的结合被计算为略有增加(而不是减少)，而天冬氨酸的结合如预期的那样被计算为强劲增加，达到与天冬氨酸结合相同的水平。成分分析提供了对这些变化的根源的洞察。双突变(K198L、D233E)具有相似的作用，而三重突变(K198L、Q199E、D233E)显著降低天冬氨酸结合。没有结合测量可用，但已知的三个突变体没有能力腺化ASN，尽管“天冬氨酸样”结合亲和力在这里计算。在对所有三个突变体的分子动力学模拟中，ASN配体的主干与实验中的Asp：ASprs复合体相比移动了1-2埃，并在口袋周围发生了显著的侧链重排。这些都可能会降低ATP结合常数和/或腺基化反应速度，解释了这些化合物缺乏催化活性的原因。最后，考虑了中性K198或带电H449与ASPR的结合，结果表明，与分析中使用的带电K198和中性H449相比，ASN与ASPR的结合不那么有利。(C)2001年学术出版社。
Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessary for correct translation of the genetic code. Engineering a modified specificity into aminoacyl-tRNA synthetases has been proposed as a means to incorporate artificial amino acid residues into proteins in vine. In a previous paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and the analogue Asn were compared by molecular dynamics free energy simulations. Molecular dynamics combined with Poisson-Boltzmann free energy calculations represent a less expensive approach, suitable for examining multiple active site mutations in an engineering effort. Here, Poisson-Boltzmann free energy calculations for aspartyl-tRNA synthetase are first validated by their ability to reproduce selected molecular dynamics binding free energy differences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase. A component analysis of the Poisson-Boltzmann free energies is employed to identify specific interactions that determine the binding affinities. The combined use of molecular dynamics free energy simulations to study one binding process thoroughly, followed by molecular dynamics and Poisson-Boltzmann free energy calculations to study a series of related ligands or mutations is proposed as a paradigm for protein or ligand design.The binding of Asn in an alternate, "head-to-tail" orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stable than the "Asp-like" orientation studied earlier. The new orientation is probably unsuitable for catalysis. A conserved active site lysine (Lys198 in Escherichia coli) that recognizes the Asp side-chain is changed to a leucine residue, found at the corresponding position in asparaginyl-tRNA synthetase. It is interesting that the binding of Asp is calculated to increase slightly (rather than to decrease), while that of Asn is calculated, as expected, to increase strongly, to the same level as Asp binding. Insight into the origin of these changes is provided by the component analyses. The double mutation (K198L,D233E) has similar effect, while the triple mutation (K198L,Q199E,D233E) reduces Asp binding strongly. No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the "Asp-like" binding affinities calculated here. in molecular dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1-2 Angstrom compared to the experimental Asp:AspRS complex, and significant side-chain rearrangements occur around the pocket. These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes. Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis. (C) 2001 Academic Press.