Investigations of the Mechanistic Pathways of the Hepatitis Delta Virus Ribozyme

Open Access
Author:
Thaplyal, Pallavi
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 26, 2014
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Chair
  • Squire J Booker, Committee Member
  • Lasse Jensen, Committee Member
  • Katsuhiko Murakami, Special Member
Keywords:
  • Ribozyme
  • Kinetics
  • HDV
  • pKa
  • QM/MM
  • Thio Effects
  • Metal Ion Rescue
Abstract:
The hepatitis delta virus (HDV) ribozyme is unique it its mechanism, as it employs both nucleobase and metal ion in its catalytic mechanism. The functional role of cytosine 75 (C75) has been established as that of a general acid based on kinetic assays, mutagenic assays, Raman spectroscopy, and x ray crystallographic studies. Recent biochemical, crystallographic, and computational studies have identified a catalytic metal ion at the active site and indicate the functional role of the metal ion in the mechanism. The most recent crystal structure of the ribozyme implicated the metal ion in multiple interactions at the active site, including interactions with the scissile phosphate. The disorder at the active site required the scissile phosphate to be modeled in from the active site of the hammerhead ribozyme, thereby raising questions about the existence and the nature of the interactions in solution. In this thesis, the focus is on the various interactions of the catalytic metal ion, the scissile phosphate, and C75 at the active site. In addition to investigating and identifying the interactions, the thesis also focuses on the catalytic mechanism, and the effect of metal ions on the reaction pathway. The focus of Chapter 2 is the interactions of the scissile phosphate at the active site. In particular, the interactions of the non-bridging (pro-RP and pro-SP) oxygen atoms are discussed. The assays utilized for the study include phosphorothioate substitution followed by metal ion rescue with thiophilic metal ion. The replacement of the pro-SP oxygen atom with sulfur was found to have no effect on the rate, whereas the replacement of the pro-RP oxygen atom with sulfur was found to decrease the rate considerably (~1000-fold, for the slower phase), in comparison to the wild-type oxo substrate. The subsequent metal ion rescue with Cd2+ was found to be stereospecific for the RP substrate, implicating a direct interaction between the metal ion and the pro-RP oxygen. The study also investigated the interaction between the pro-RP oxygen and C75, and found that the interaction may be redundant in the presence of multitude of interactions at the active site. This work is not only a fundamental biochemical study relating structure-function in ribozymes, but also an example of the significance of complementing crystallographic data with biochemical studies to obtain a comprehensive picture of intricate reaction mechanisms. The focus of Chapter 3 is determining the mechanistic pathway of the HDV ribozyme catalysis using theoretical calculations (quantum mechanics/molecular mechanics- QM/MM) complemented by biochemical assays. QM/MM simulations showed unique pathways in the presence of monovalent (Na+) ions alone versus divalent metal (Mg2+) ions, which agree well with earlier experimental findings from our lab. The energy barrier obtained in the presence of Na+ was found to be lower than that in the presence of Mg2+, which contradicted experimental observations. Thus, further experimental studies were conducted in an effort to resolve the discrepancy in the theoretically calculated energy barriers. The biochemical experiments were designed to compare the pKa of 2’OH in RNA in the presence of monovalent and divalent metal ions, since the energetic contribution of 2’OH deprotonation was not accounted for in the theoretical calculations. The pKa of the 2’OH was determined using two different approaches: kinetic and NMR, and both showed that divalent metal ions lower the pKa of 2’OH to favor deprotonation as compared to monovalent metal ions. The focus of Chapter 4 is utilizing inverse thio effects as a general mechanistic diagnostic in ribozyme catalyzed reactions. As mentioned before, QM/MM simulations have shown that the HDV ribozyme reaction goes via a sequential mechanism through a phosphorane intermediate in the presence of Na+, and a concerted mechanism through a transition state in the presence of Mg2+. In addition, preliminary studies conducted in the presence of Na+, showed a normal thio effect for the RP substrate, but an inverse thio effect for the SP substrate. The inverse thio effect was a unique stereospecific feature, and further studies were conducted to understand the origin of the effect. In the presence of both monovalent and divalent metal ions in group I and IIA, larger diffuse metal ions were found to show an inverse thio effect (Na+: 5-fold, K+: 6-fold, Ba2+: 3-fold). To probe the possible link between the inverse thio effect and sequential mechanistic pathway (scenario 1), a model oligonucleotide cleavage assay was used that is known to go via a sequential mechanism. Inverse thio effects were observed for the model oligonucleotide albeit the effects were subtle (2-fold), and hence the results were not able to provide conclusive evidence. In addition, classical molecular dynamics (MD) studies were also performed to investigate a new inhibitory interaction between the 2’OH and the pro-SP oxygen atom (scenario 2). The interaction was found to exist only in the presence of Na+. Thus, the loss of the inhibitory interaction between the 2’OH and the pro-SP atom in the case of the SP substrate may give rise to the inverse thio effect in the presence of Na+. To distinguish and identify the scenario that results in the inverse thio effect, future experiments and calculations are also discussed. Overall the work presented in this thesis, provides evidence for certain key interactions involving the scissile phosphate and attempts to provide a general diagnostic for the mechanistic pathway in ribozymes.