A combined experimental and theoretical study of divalent metal ion selectivity and function in proteins: Application to E-coli ribonuclease H1
A combined experimental and theoretical study of divalent metal ion selectivity and function in proteins: Application to E-coli ribonuclease H1
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DOI：
10.1021/ja034956w
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发表时间：
2003-08-06
影响因子：
15
通讯作者：
Lim, C
中科院分区：
文献类型：
--
作者：
Babu, CS;Dudev, T;Lim, C
用实验和理论相结合的方法研究了碱土金属离子(镁、钙、锶、Ba2+)与大肠杆菌核糖核酸酶H1(RNaseH1)结合的结构和热力学性质。用经典的分子动力学模拟研究了酶的各种金属结合模式，并用自由能模拟计算了酶的相对结合自由能。随后结合密度泛函理论和连续介质介电方法验证了基于模拟结构的模型体系自由能的变化趋势。这些计算为实验结果提供了物理基础，并提出了金属阳离子和催化重要的酸性残基在蛋白质功能中的合理作用(S)。镁离子通过其四个第一壳层水分子释放活性中心羧酸盐残基之一D70作为一般碱，从而间接激活磷原子的水攻击，阻止D70直接与镁离子结合。另一方面，钙离子通过与D70的两个羧基氧原子发生双齿相互作用，阻止D70作为一般碱而抑制酶的活性。这些对D70的额外作用，以及对镁离子的D10和E48单齿相互作用，使钙离子比其他二价离子更紧密地结合在一起。然而，在第一壳中含有两个或更少水分子的裸露的镁离子可以直接与三个活性部位的羧酸盐结合，特别是D70，从而抑制酶的活性。本文的分析和结果可以推广到RNaseH家族的其他成员，这些成员具有相同的结构折叠，并显示出类似的金属结合部位和依赖于镁的活性。
Structural and thermodynamic aspects of alkaline earth metal dication (Mg2+, Ca2+, Sr2+, Ba2+) binding to E coli ribonuclease H1 (RNase H1) have been investigated using both experimental and theoretical methods. The various metal-binding modes of the enzyme were explored using classical molecular dynamics simulations, and relative binding free energies were subsequently evaluated by free energy simulations. The trends in the free energies of model systems based on the simulation structures were subsequently verified using a combination of density functional theory and continuum dielectric methods. The calculations provide a physical basis for the experimental results and suggest plausible role(s) for the metal cation and the catalytically important acidic residues in protein function. Magnesium ion indirectly activates water attack of the phosphorus atom by freeing one of the active site carboxylate residues, D70, to act as a general base through its four first-shell water molecules, which prevent D70 from binding directly to Mg2+. Calcium ion, on the other hand, inhibits enzyme activity by preventing D70 from acting as a general base through bidentate interactions with both carboxylate oxygen atoms of D70. These additional interactions to D70, in addition to the D10 and E48 monodentate interactions found for Mg2+, enable Ca2+ to bind tighter than the other divalent ions. However, a bare Mg2+ ion with two or less water molecules in the first shell could bind directly to the three active-site carboxylates, in particular D70, thus inhibiting enzymatic activity. The present analyses and results could be generalized to other members of the RNase H family that possess the same structural fold and show similar metal-binding site and Mg2+-dependent activity.