Computational studies of the structure, dynamics, and catalysis of the hepatitis delta virus ribozyme

Ribozymes represent a unique class of RNA that is capable of catalyzing biochemical reactions. Since their discovery three decades ago, ribozymes have been shown to be involved in numerous crucial biological processes, such as gene regulation, translation, and RNA splicing. Similar to protein enzymes, these catalytic RNAs fold into well-defined tertiary structures with organized active sites to carry out catalysis. While their side chains do not possess the variety in functional groups accessible to the side chains of their protein counterparts, their efficiency as biological catalysts is often comparable to protein enzymes, making their catalytic strategies highly interesting. The study of ribozymes is also motivated by their possible significance in evolutionary biology. The hepatitis delta virus (HDV) ribozyme is a small nucleolytic RNA that performs a phosphodiester self-cleavage reaction generating products with a 2’, 3’-cyclic phosphate and a 5’ -hydroxyl termini. This ribozyme was initially discovered in the RNA genome of the human pathogen HDV, where it played a crucial role in the viral life cycle, cleaving site-specifically the multimeric copies of the RNA genome into monomeric pieces. More recently, HDV-like ribozymes have been shown to be widespread across all kingdoms of life. Since their discovery, the HDV ribozymes have been intensely studied from structural as well as mechanistic perspectives, and much is known about their structure and catalytic strategies. The HDV ribozyme has a compact double-pseudoknot structure and uses a combination of metal ion and nucleobase catalysis to effect its self-cleavage reaction. An active site cytosine C75 is thought to act as a general acid in the catalytic reaction by donating a proton to the 5’-hydroxyl of the leaving group, and an active site Mg2+ ion has been purported to play the role of a Lewis acid and activate the O2’ nucleophile. The exact role of this putative catalytic ion is still uncertain. A crystal structure also revealed a rare reverse G•U wobble close to the active site interacting with the putative catalytic ion. This base pair has been hypothesized to play an important role in positioning the metal ion for catalysis. In this dissertation, the HDV ribozyme was studied using a variety of computational approaches. Classical molecular dynamics (MD) simulations and non-linear Poisson-Boltzmann (NLPB) calculations were utilized to study the metal binding characteristics of the reverse G•U wobble close to the active site of the ribozyme. These studies revealed that the reverse wobble creates a highly negative pocket that allows it to interact with metal ions and helps to shift the pKa of the nucleobase C75, thereby facilitating its protonation. MD simulations were also used to investigate the impact of C75 protonation and Mg2+ ion interaction at the reverse G•U wobble on the structure as well as the motions of the HDV ribozyme. The protonated state of C75 was found to be essential for keeping the active site organized for catalysis. A localized, ‘chelated’ metal ion interaction was observed at the reverse G•U wobble, in contrast to a ‘diffused’ metal ion interaction observed at a standard G•U wobble also located close to the active site. The effects of mutation of the reverse G•U wobble, as well as the standard G•U wobble, to a Watson-Crick GC base pair were also studied. The overall tertiary structure and thermal motions of the ribozyme were not found to be significantly affected by C75 protonation, mutation of the reverse and standard wobbles, or the metal ion interaction at the two wobbles, suggesting that small local motions at the active site, rather than large-scale global motions, dominate the ribozyme reaction pathway. Quantum mechanical/molecular mechanical (QM/MM) calculations were used to study the HDV ribozyme self-cleavage reaction and elucidate the role of the catalytic metal ion. The calculations suggested a concerted mechanism of the catalytic reaction in the presence of a divalent ion at the active site but a sequential mechanism in the presence of a monovalent ion at the same position. The divalent ion at the active site was found to lower the pKa of the nucleobase C75, making its proton donation more facile and thereby favoring the concerted mechanism. QM/MM calculations were also used to study the effects of phosphorothioate substitutions of the non-bridging oxygens at the scissile phosphate, commonly known as the ‘thio effects’, in the HDV ribozyme. In the case of the RP sulfur substrate, the calculations revealed a reactant state with pronounced active site distortion and an unfavorable reaction pathway with a high energetic barrier. In contrast, the reactant state of the SP sulfur substrate showed minimal distortion at the active site compared to the oxo-substrate. The results from these studies are consistent with several biochemical experimental studies. The mechanism of the HDV ribozyme was further investigated using QM/MM free energy simulations to include conformational sampling and entropic effects. Umbrella sampling simulations were combined with a finite temperature string method to generate the multidimensional free energy surface underlying the self-cleavage reaction. The results of these simulations were qualitatively consistent with the previous QM/MM calculations, indicating a concerted mechanism in the presence of a Mg2+ ion at the catalytic site and a sequential mechanism in the presence of a Na+ ion. However, several new mechanistic insights were provided by the QM/MM free energy simulations, including the observation of proton transfer from the exocyclic amine of protonated C75 to the nonbridging oxygen of the scissile phosphate to stabilize the phosphorane intermediate in the sequential mechanism. The free energy barrier along the concerted pathway in the presence of the catalytic Mg2+ ion was consistent with the intrinsic reaction rate of the HDV ribozyme cleavage reaction measured experimentally. The differences in the reaction pathways of the cleavage reaction in the presence of the Mg2+ ion and the Na+ ion illustrated several key roles of the catalytic metal ion in the HDV ribozyme catalysis, including activation of the O2’ nucleophile, acidification of the general acid C75, and stabilization of the non-bridging oxygen of the scissile phosphate