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

摘要

The hepatitis delta virus (HDV) ribozyme usesnboth metal ion and nucleobase catalysis in its cleavage mechan-nism. A reverse G3nU wobble was observed in a recent crystalnstructure of the precleaved state. This unusual base pair posi-ntions a Mgn2þ ion to participate in catalysis. Herein, we usednmolecular dynamics (MD) and X-ray crystallography to char-nacterize the conformation and metal binding characteristics ofnthis base pair in product and precleaved forms. Beginning with ancrystal structure of the product form, we observed formation ofnthe reverse G3nU wobble during MD trajectories. We alsondemonstrated that this base pair is compatible with the diffraction data for the product-bound state. During MD trajectories ofnthe product form, Nanþ ions interacted with the reverse G3nU wobble in the RNA active site, and a Mgn2þ ion, introduced in certainntrajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G3nU wobble withnbound Mgn2þ remained intact during MD simulations. When we removed Mgn2þ from the starting precleaved structure, Nanþ ionsninteracted with the reverse G3nU wobble. In support of the computational results, we observed competition between Nanþ andMgn2þnin the precleaved ribozyme crystallographically. Nonlinear Poisson-Boltzmann calculations revealed a negatively charged patchnnear the reverse G3nU wobble. This anionic pocket likely serves to bind metal ions and to help shift the pKa of the catalyticnnucleobase, C75. Thus, the reverse G3nU wobble motif serves to organize two catalytic elements, a metal ion and catalyticnnucleobase, within the active site of the HDV ribozyme.