Archontis G, Simonson T, Moras D, Karplus M
Laboratoire de Chimie Biophysique, Institut Le Bel, Université Louis Pasteur, 4 rue Blaise Pascal, Strasbourg, 67000, France.
J Mol Biol. 1998 Feb 6;275(5):823-46. doi: 10.1006/jmbi.1997.1470.
Specific amino acid binding by aminoacyl-tRNA synthetases is necessary for correct translation of the genetic code. To obtain insight into the origin of the specificity, the binding to aspartyl-tRNA synthetase (AspRS) of the negatively charged substrate aspartic acid and the neutral analogue asparagine are compared by use of molecular dynamics and free energy simulations. Simulations of the Asn-AspRS complex show that although Asn cannot bind in the same position as Asp, several possible positions exist 1.5 to 2 A away from the Asp site. The binding free energy of Asn in three of these positions was compared to that of Asp through alchemical free energy simulations, in which Asp is gradually mutated ito Asn in the complex with the enzyme. To correctly account for the electrostatic interactions in the system (including bulk solvent), a recently developed hybrid approach was used, in which the region of the mutation site is treated microscopically, whereas distant protein and solvent are treated by continuum electrostatics. Seven free energy simulations were performed in the protein and two in solution. The various Asn positions and orientations sampled at the Asn endpoints of the protein simulations yielded very similar free energy differences. The calculated Asp-->Asn free energy change is 79.8(+/-1.5) kcal/mol in solution and 95.1(+/-2.8) kcal/mol in the complex with the protein. Thus, the substrate Asp is predicted to bind much more strongly than Asn, with a binding free energy difference of 15.3 kcal/mol. This implies that erroneous binding of Asn by AspRS is highly improbable, and cannot account for any errors in the translation of the genetic code. Almost all of the protein contributions to the Asp versus Asn binding free energy difference arise from an arginine and a lysine residue that hydrogen bond to the substrate carboxylate group and an Asp and a Glu that hydrogen bond to these; all four amino acid residues are completely conserved in AspRSs. The protein effectively "solvates" the Asp side-chain more strongly than water does. The simulations are analyzed to determine the interactions that Asn is able to make in the binding pocket, and which sequence differences between AspRS and the highly homologous AsnRS are important for modifying the amino acid specificity. A double or triple mutation of AspRS that could make it specific for Asn is proposed, and supported by preliminary simulations of a mutant complex.
氨酰 - tRNA合成酶对特定氨基酸的结合对于遗传密码的正确翻译是必要的。为了深入了解特异性的起源，通过分子动力学和自由能模拟比较了带负电荷的底物天冬氨酸和中性类似物天冬酰胺与天冬氨酰 - tRNA合成酶（AspRS）的结合情况。天冬酰胺 - AspRS复合物的模拟表明，虽然天冬酰胺不能与天冬氨酸结合在相同位置，但在距离天冬氨酸位点1.5至2埃处存在几个可能的位置。通过炼金术自由能模拟将天冬酰胺在其中三个位置的结合自由能与天冬氨酸的结合自由能进行比较，在该模拟中，天冬氨酸在与酶的复合物中逐渐突变为天冬酰胺。为了正确考虑系统中的静电相互作用（包括本体溶剂），使用了一种最近开发的混合方法，其中突变位点区域进行微观处理，而远处的蛋白质和溶剂则通过连续介质静电学处理。在蛋白质中进行了七次自由能模拟，在溶液中进行了两次。在蛋白质模拟的天冬酰胺端点处采样的各种天冬酰胺位置和取向产生了非常相似的自由能差异。计算得出在溶液中天冬氨酸向天冬酰胺的自由能变化为79.8（±1.5）千卡/摩尔，在与蛋白质的复合物中为95.1（±2.8）千卡/摩尔。因此，预计底物天冬氨酸的结合比天冬酰胺强得多，结合自由能差为15.3千卡/摩尔。这意味着AspRS错误结合天冬酰胺的可能性极小，并且不能解释遗传密码翻译中的任何错误。几乎所有蛋白质对天冬氨酸与天冬酰胺结合自由能差异的贡献都来自与底物羧酸盐基团形成氢键的一个精氨酸和一个赖氨酸残基，以及与这些残基形成氢键的一个天冬氨酸和一个谷氨酸；所有四个氨基酸残基在天冬氨酰 - tRNA合成酶中完全保守。蛋白质比水更有效地“溶剂化”天冬氨酸侧链。对模拟进行分析以确定天冬酰胺在结合口袋中能够形成的相互作用，以及天冬氨酰 - tRNA合成酶与高度同源的天冬酰胺 - tRNA合成酶之间的哪些序列差异对于改变氨基酸特异性很重要。提出了一种可能使其对天冬酰胺具有特异性的AspRS双突变或三突变，并得到了突变复合物初步模拟的支持。
