Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: does a third metal binding site modulate endonuclease catalysis?

Abstract

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg(2+) concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg(2+) concentration. The catalytic center is known to contain two Mg(2+) ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg(2+). Importantly, the presence of a third Mg(2+) ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg(2+). At standard concentration, a third Mg(2+) is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg(2+) ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg(2+) ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg(2+) ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.

Figure 1. Crystallographic structure of Bacillus halodurans RNase H

(A) Cartoon of the complex of RNase H and ds-RNA/DNA hybrid duplex. DNA and RNA are drawn in red and blue ribbons, respectively; orange spheres indicate the Mg²⁺ ions. (B) Close-up of the catalytic site, including the RNA strand, metal ligands and water molecules coordinated to the two crystallographic Mg²⁺ ions. The key residue E188 should be noted. The D192N inactive mutant is shown (PDB code: 1ZBL3).

Figure 2. Catalytic mechanism of RNase H as proposed by QM/MM calculations

R: reactants state, the nucleophilic water (Wat_N) deprotonated in situ is represented in red, and the water H-bond network promoting the proton transfer to the phosphate group is shown in blue. TS₁-INT-TS₂: composite transition states and high-energy intermediate as calculated in previous studies. P: product state of the cleaved RNA strand and its release from the metal site.

Figure 3. Active and inactive states as modulated by MgCl₂ concentration

Two representative snapshots taken from the MD simulations represent the active state (A) and the inactive state (B) dominant, respectively, at low (25 mM) and high (500 mM) Mg²⁺ concentration. (A) Active state: the carboxylate group of E188 points out of the active site and Wat_N binds to MgA. (B) Inactive state: Wat_N coordinates to MgC and slightly loses contact to MgA, while the carboxylate group of E188 flips inward and points to the phosphodiester group. Metal-ligand coordination is indicated with dashed lines.

Figure 4. Mg²⁺ distribution at the active site of RNase H

The number of coordinated Mg²⁺ ions (NC) around the C5 phosphodiester P atom is shown as extracted from the MD simulations of wild-type (A) and E188A mutated system (B) at different MgCl₂ concentration. NC(r) is derived from the integration of the P-Mg²⁺ radial distribution function. The localization of MgC in the active and inactive state is shown by the blue and red areas, respectively (Figure 3).

Figure 5. Free energy landscape of the active site architecture at low and high MgCl₂ concentration

The free energy surface at 25 mM (red) and 500 mM (blue) of MgCl₂ as a function of the MgC to phosphodiester group phosphorus atom (P) separation, RC₁ (A); the MgA-Wat_N separation, RC₂ (B); and E188 to phosphodiester group distance, RC₃ (C). The reaction coordinate RC₃ is chosen as the distance that separates the carboxylate group (C_δ) of E188 and the phosphorus atom (P). Insets represent graphically the reaction coordinates adopted in ABF calculations.

Figure 6. Calculated electrostatic properties of the active site in RNase H

Shown are electrostatic potentials (ESP) obtained using the Adaptive Poisson-Boltzmann Solver method with dielectric constants of 1.0 and 78.5 for the solute and solvent, respectively (color scale: +10 kT/e (blue) and −10 kT/e (red)). The RNA/DNA hybrid is represented as a yellow ribbon. The calculated ESP for the wild-type system (A) and for the E188A mutant (B) is mapped on the protein molecular surface. Position 188 in both the systems is indicated by residue labels; the orange sphere represents the location of the third Mg²⁺ ion.

Figure 7. Active sites of other members of the NT superfamily showing a second-shell glutamate residue

(A) The active conformation from the present work on Bh RNase H; (B) E. coli RNase H X-ray structure (pdb code: 1g15); (C) prokaryotic DNA transposase (pdb code: 1mus); (D) Aa-RNase III/ds-RNA complex (pdb code: 2nug). HIV reverse transcriptase (pdb code: 1suq) also shows this motif (structure not shown). Magnesium ions are represented as orange spheres, manganese ions as green spheres. Divalent ions that superimpose to MgA, MgB, MgC in the present work are labeled specifically.