45534-AC4
Water as a Specific Acid/Base Catalysts in the Mechanism of Action of a Non-Coding Catalytic RNA
Ribozymes are naturally occurring RNA enzymes that catalyze a diverse number of biological reactions such as protein synthesis, intron splicing and tRNA processing. To elucidate the principles of ribozyme catalysis, the Wedekind lab studies the hairpin ribozyme. During the course of recent crystallographic investigations, we detected ordered water molecules within a solvent sequestered pocket. Due to the limited functional group repertoire of RNA enzymes, it has been hypothesized that such water molecules participate directly in the reaction by serving as specific acid or base catalysts, or in a proton relay.
The goal of this research is to identify and assess the role of key waters in the hairpin ribozyme active site. The significance of each water molecule will be assessed by its location in crystal structures representative of pre-catalytic, reaction-intermediate and product-bound states (Aim 1). Subsequently, a series of modified, purine nucleosides will be synthesized and incorporated into the active site to displace specific waters. These ribozymes will be subjected to single-turnover activity assays and analyzed for the ability to adopt the native fold (Aim 2).
Progress was made on both aims during the award period (09-01-06 to 08-31-08). In the first year we developed a minimal, hinged hairpin ribozyme crystallization construct with superior folding properties (MacElrevey et al. 2007). Structures of these hinged molecules were solved the presence of: (i) vanadium (a transition-state analog), and (ii) a product mimic comprising cis diols at A-1 with a free 5'-OH group at G+1 (Torelli et al. 2007). This work advanced our knowledge of the reaction-coordinate conformation and provided compelling evidence that water molecules promote the transition-state by contributing electrostatic stabilization (Aim 1). To improve our understanding of the role of water in the product-bound state, we completed synthesis of the novel nucleoside analog, 5'-deoxy-5'-flouro-guanosine or 5FG (Spitale et al. 2007). In the past year, we advanced our understanding of the product-bound state by incorporation of 5FG into the hairpin ribozyme at G+1, which when combined with cis diols at A-1 produced high-quality crystals. This product mimic, in which fluorine substitutes for the 2'-nucleophile, provided proof-of-principle to proceed with crystallization of the 5FG adduct in complex with the bona fide 2',3'-cyclic phosphate product. This work is ongoing and should yield detailed information on the location of candidate, specific-base water molecules in the pre-ligation state.
Significant progress on Aim 2 was realized in the past year. We synthesized a novel N1-purine diversity library utilizing the chemistry of Herdewijn. The six-nucleoside library comprises N1-alkyl and N1-alkanol substituents on an adenosine platform. However, during library generation it became clear we would become constrained by time and cost. Therefore, future efforts will focus on the use of Dimroth conditions to promote N1-to-N6-alkyl rearrangement in the context of RNA to increase library diversity. In the meantime, we synthesized N1-deaza-adenosine, as reported by Cristalli, and completed the novel synthesis of N1-deaza-guanosine. We incorporated N1-deaza-adenosine into the hairpin ribozyme at positions A9, A10 and A38, respectively. The A38 variant was catalytically inactive, whereas A9 and A10 had modest effects implying they are not essential to position 'catalytic' waters. Crystallographic analysis of A38 in the pre-catalytic state revealed the global ribozyme fold is intact, but subtle conformational changes exist at the active site. These observations highlight the importance of the N1 moiety of A38 in chemistry and demonstrate that loss of catalytic activity is not due to a fold defect. At present, the precise role of A38 in catalysis remains unknown. Importantly, our structural work provides no evidence that water binds at A38 N1. Moreover, our kinetic analyses suggest that if water serves as a specific acid, it is incapable of effective function without A38 N1. These topics, including a complete analysis of N1-deaza-guanine at position G8, are topics for future investigation.
Support from the PRF provided valuable training experience for several students. Mr. Robert Spitale, whose stipend was paid by PRF, conducted the synthesis, kinetic assays and structure determinations of this project. He earned an M.S. degree during the grant period, and will complete his doctorate in chemistry by mid-2010. Three undergraduates were trained including: Moriah Heller, who graduated with a B.S. in biochemistry and is currently a graduate student at Sloan-Kettering. Amanda Pelly was an REU fellow who graduated with a B.S. from Cornell and currently works in the healthcare field. Michael Mungillo is a chemistry undergraduate who started on this project as an REU student and will remain in the lab to finish analyses of N1-deaza-G8. This project has enriched the research and training environment for two doctoral students, Andrew Torelli and Celeste MacElrevey. These students were not directly supported by PRF, but benefited from interactions with the aforementioned personnel. Overall, support from the PRF provided a platform to launch two, expanded research proposals to the NIH (in review October 2009).
