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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• 作者：C. Satheesan Babu ; Todor Dudev ; R. Casareno ; J. A. Cowan ; and Carmay Lim
• 刊名：Journal of the American Chemical Society
• 出版年：2003
• 出版时间：August 6, 2003
• 年：2003
• 卷：125
• 期：31
• 页码：9318 - 9328
• 全文大小：496K
• 年卷期：v.125,no.31(August 6, 2003)
• ISSN：1520-5126

文摘

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 andtheoretical methods. The various metal-binding modes of the enzyme were explored using classicalmolecular dynamics simulations, and relative binding free energies were subsequently evaluated by freeenergy simulations. The trends in the free energies of model systems based on the simulation structureswere subsequently verified using a combination of density functional theory and continuum dielectricmethods. 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 ionindirectly 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 bindingdirectly to Mg²⁺. Calcium ion, on the other hand, inhibits enzyme activity by preventing D70 from acting asa general base through bidentate interactions with both carboxylate oxygen atoms of D70. These additionalinteractions 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 inthe first shell could bind directly to the three active-site carboxylates, in particular D70, thus inhibitingenzymatic activity. The present analyses and results could be generalized to other members of the RNaseH family that possess the same structural fold and show similar metal-binding site and Mg²⁺-dependentactivity.