The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pK(a) shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg(2+) ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg(2+) ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg(2+) ion is also found within the active site where it interacts with a newly resolved G.U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-R(P) oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg(2+) ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

The hepatitis delta virus (HDV) ribozyme uses a cytosine to facilitate general acid-base catalysis. Biochemical studies suggest that C75 has a pKa perturbed to near neutrality. To measure this pKa directly, Raman spectra were recorded on single ribozyme crystals using a Raman microscope. A spectral feature arising from a single neutral cytosine was identified at 1528 cm(-1). At low pH, this mode was replaced with a new spectral feature. Monitoring these features as a function of pH revealed pKa values for the cytosine that couple anticooperatively with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These pKa values agree well with those obtained from ribozyme activity experiments in solution. To correlate the observed pKa with a specific nucleotide, crystals of C75U, which is catalytically inactive, were examined. The Raman difference spectra show that this mutation does not affect the conformation of the ribozyme. However, crystals of C75U did not produce a signal from a protonatable cytosine, providing strong evidence that protonation of C75 is being monitored in the wild-type ribozyme. These studies provide the first direct physical measurement of a pKa near neutrality for a catalytic residue in a ribozyme and show that ribozymes, like their protein enzyme counterparts, can optimize the pKa of their side chains for proton transfer.

The hepatitis delta virus (HDV) ribozyme uses both metal ion and nucleobase catalysis in its cleavage mechanism. A reverse G•U wobble was observed in a recent crystal structure of the precleaved state. This unusual base pair positions a Mg 2+ 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•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•U wobble in the RNA active site, and a Mg 2+ ion, introduced in certain trajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G•U wobble with bound Mg 2+ remained intact during MD simulations. When we removed Mg 2+ from the starting precleaved structure, Na + ions interacted with the reverse G•U wobble. In support of the computational results, we observed competition between Na + and Mg 2+ in the precleaved ribozyme crystallographically. Non-linear Poisson-Boltzmann calculations revealed a negatively charged patch near the reverse G•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•U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme.RNA is involved in many aspects of biology, where it serves both informational and functional roles (1-3). Indeed, RNA can act as a riboswitch, binding small molecules and † This project was supported by NIH grant R01GM095923 (B.L.G and P.C.B), NIH grant GM56207 (S.H.S.), instrumentation funded by the NSF through grant OCI-0821527, the Purdue University Department of Biochemistry, the Markey Center for Structural Biology and the Purdue University Center for Cancer Research (B.L.G.). * To whom correspondence should be addressed. B.L.G.: telephone (765) 496-6165; fax (765) 494-7897; barbgolden@purdue.edu. S.H.-S. telephone (814) 865-6442; fax (814) 865-2927; shs@chem.psu.edu. P.C.B. telephone (814) 863-3812; fax (814) 865-2927. pcb@chem.psu.edu. ⊥ Present Address: Department of Biochemistry, University of Illinois, 600 S. Mathews Ave., Urbana IL 61801.Supporting Information Available Procedures for MD; protonated cytosine partial charges; additional MD trajectories; metal ion movement during MD; heavy-atom RMSD plots; additional NLPB results. Also provided are tables of crystallographic data collection statistics; crystallographic Na + coordinates for the pre-cleaved ribozyme; and average distances between the reverse G•U wobble and metal ions from MD. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBioche...

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted > 2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335–13342.], while in crystal structures, it is well-positioned for proton transfer. However no crystallographic evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on rate and Mg2+-binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously-unobserved hydrated magnesium ion interacts with the N7 of the cleavage site G•U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.