Metal Binding Motif in the Active Site of the HDV Ribozyme Binds Divalent and Monovalent Ions

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• 作者：Narayanan Veeraraghavan ; Abir Ganguly ; Jui-Hui Chen ; Philip C. Bevilacqua ; Sharon Hammes-Schiffer ; Barbara L. Golden
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：April 5, 2011
• 年：2011
• 卷：50
• 期：13
• 页码：2672-2682
• 全文大小：1021K
• 年卷期：v.50,no.13(April 5, 2011)
• ISSN：1520-4995

文摘

The hepatitis delta virus (HDV) ribozyme uses both metal ion and nucleobase catalysis in its cleavage mechanism. A reverse G路U wobble was observed in a recent crystal structure of the precleaved state. This unusual base pair positions a Mg²⁺ ion to participate in catalysis. Herein, we used molecular dynamics (MD) and X-ray crystallography to characterize the conformation and metal binding characteristics of this base pair in product and precleaved forms. Beginning with a crystal structure of the product form, we observed formation of the reverse G路U wobble during MD trajectories. We also demonstrated that this base pair is compatible with the diffraction data for the product-bound state. During MD trajectories of the product form, Na⁺ ions interacted with the reverse G路U wobble in the RNA active site, and a Mg²⁺ ion, introduced in certain trajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G路U wobble with bound Mg²⁺ remained intact during MD simulations. When we removed Mg²⁺ from the starting precleaved structure, Na⁺ ions interacted with the reverse G路U wobble. In support of the computational results, we observed competition between Na⁺ and Mg²⁺ in the precleaved ribozyme crystallographically. Nonlinear Poisson鈭払oltzmann calculations revealed a negatively charged patch near the reverse G路U wobble. This anionic pocket likely serves to bind metal ions and to help shift the pK_a of the catalytic nucleobase, C75. Thus, the reverse G路U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme.