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

Abstract

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 pK_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 pK_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.

Figure 1

Illustration of the active site interactions in HHR and its self-cleavage mechanism. The N1 position of guanine in the putative general base G12 (blue) needs to be deprotonated before acting as a proton acceptor to deprotonate the 2′-OH in C17 (red), which will then act as the nucleophile to attack the phosphorous. Recent studies(15, 16) indicate that there could be a Mg²⁺ directly bound at the Hoogsteen face of G12 (“G-site”) to facilitate its deprotonation. Another Mg²⁺ is believed to play the role of activating the 2′-OH of the general acid G8 (green) by migrating from the binding site at N7 of G10.1 (“C-site”) observed crystallographically into a bridging position (“B-site”) with the scissile phosphate, in accord with thio/rescue effect experiments(29, 39, 57). In this bridging position, the Mg²⁺ can coordinate the 2′-OH of G8, increasing its acidity, and facilitating proton transfer to the O5′ leaving group in the general acid step of the reaction(8).

Figure 2

(Left) Canonical numbering of guanine nucleobase. (Right) Resonance structures of guanine deprotonated at N1 position with formal charge alternately on the N1 and the O6.

Figure 3

Chemical structures of guanine and several chemically modified guanine molecules studied in this work. Experimental pK_a values at the N1 position (taken from Refs. 90, 91) are shown.

Figure 4

Optimized geometries of Mg²⁺-guanine (A) and Mg²⁺-deprotonated guanine (B) complexes. Selected bond lengths shown are in A.

Figure 5

Thermodynamic cycle used in TI calculations.

Figure 6

Convergence of pK_a shift values from TI simulations. Filled squares connected by solid lines are the pK_a shift values evaluated using all available data at certain simulation time, with an increment of 100 ps per point. Dashed lines are drawn to help show the convergence.

Figure 7

Representative active site conformation of the initial (left) and final (right) states from the free energy (TI) simulation of HHR · Mg²⁺:G12. The conformations from the simulation of HHR:G12 are very similar, and therefore not shown here. For clarity, water molecules and some other atoms/residues are not displayed. White, cyan, blue, red, pink and gold spheres stand for H, C, N, O, Mg and P atoms, respectively.

Figure 8

Radial distribution function (RDF) of water oxygens around the O6 position of neutral and deprotonated G12 in HHR. Both sets of data are extracted from 1 ns of MD simulation of HHR:G12 with G12 in neutral and deprotonated forms, respectively. RDF data points are calculated using window size of 0.2 A and are interpolated using the Akima spline.

Figure 9

Potential of mean force (PMF) for the general base proton transfer (GBPT) reaction in HHR · Mg²⁺:G12 (red) and HHR:G12 (black) generated by ab initio QM/MM umbrella sampling. The two chemical structures depict the reactant (left) and product (right) states. Reaction coordinate is the difference (R₁–R₂) between the distance from C17:O2′ to C17:HO2′ (R₁) and the distance from G12:N1 to C17:HO2′ (R₂). Free energies of the reactant and product states are marked.