Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the p<i>K</i>_A of G12. On the basis of this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, key questions remain about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straightforward interpretation of the measured activity–pH data implies the p<i>K</i>_a value of the N1 position in the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a p<i>K</i>_a shift of G12 toward neutrality. We present computational results that support this hypothesis and provide a model that unifies the interpretation of available structural and biochemical data and paints a detailed mechanistic picture of the general base step of the reaction. Experimentally testable predictions are made for mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis

Thio Effects and an Unconventional Metal Ion Rescue in the Genomic Hepatitis Delta Virus Ribozyme

Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the <i>pro-R</i>_P oxygen at the scissile phosphate directly coordinates a catalytic Mg²⁺ ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn²⁺. Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd²⁺. Reaction profiles with an <i>S</i>_P-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg²⁺ or Cd²⁺, supporting the idea that the <i>pro-S</i>_P oxygen does not coordinate metal ions. The <i>R</i>_P-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold and the slow-reacting phase displaying a thio effect of ∼1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd²⁺ of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd²⁺ inhibiting the oxo substrate but not the <i>R</i>_P substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the <i>pro-R</i>_P oxygen, in line with experiments with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg²⁺ binding, or with C75 deleted suggest that the <i>pro-R</i>_P oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the <i>pro-R</i>_P oxygen and the nucleophilic 2′-hydroxyl, supporting the experimental findings

Identification of the Catalytic Mg²⁺ Ion in the Hepatitis Delta Virus Ribozyme

The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a p<i>K</i>_a shifted toward neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the precleavage ribozyme identified a Mg²⁺ ion that interacts through its partial hydration sphere with the G25·U20 reverse wobble. In addition, this Mg²⁺ ion is in position to directly coordinate the nucleophile, the 2′-hydroxyl of U(−1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2′-hydroxyl. To test the role of the active site Mg²⁺ ion, we replaced the G25·U20 reverse wobble with an isosteric A25·C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg²⁺ binding could be adversely affected by the mutation. The kinetic analysis and molecular dynamics of the A25·C20 double mutant suggest that this variant stably folds into an active structure. However, pH–rate profiles of the double mutant in the presence of Mg²⁺ are inverted relative to the profiles for the wild-type ribozyme, suggesting that the A25·C20 double mutant has lost the active site metal ion. Overall, these studies support a model in which the partially hydrated Mg²⁺ positioned at the G25·U20 reverse wobble is catalytic and could serve as a Lewis acid, a Brønsted base, or both to facilitate deprotonation of the nucleophile

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn²⁺ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH–rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the p<i>K</i>_a of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction