Bridging the gap between theory and experiment to derive a detailed understanding of hammerhead ribozyme catalysis

Abstract

Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg(2+) ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg(2+) ion or multiple Na(+) ions, one of which is also in a bridging coordination pattern. In the case of a single Mg(2+) ion bound in the active site, the Mg(2+) ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2'). As the reaction proceeds, the Mg(2+) ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2'. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis.

Keywords: RNA; catalysis; combined QM/MM; enzyme; free energy; hammerhead ribozyme; mechanism; simulation.

Figure 2.1

The minimal functional HHR sequence (mHHR) with conserved core region labeled with bold and italic letters. The letter N represents any RNA nucleotide (under the complimentary sequence constraint).

Figure 2.2

Possible catalytic role of Mg²⁺ in the eHHR. The C-site in the prereactive state involves Mg²⁺ binding at the N7 of G10.1 and the A9 pro-R phosphate oxygen. Activation of the 2′OH may occur through interactions with G12, the proposed general base. Migration from the C-site to a bridging position between A9 and the scissile phosphates occurs in proceeding to the transition state in which the Mg²⁺ acquires additional interaction with the O5′ leaving group and the 2′OH of G8, the implicated general acid.

Figure 2.3

The Mg²⁺ positions from snapshots of simulations with Mg²⁺ initially placed at the C-site position. Snapshots shown are for the initial C-site position (upper left), the reactant state with C17:O2′ protonated (upper right), the reactant state with C17:O2′ deprotonated (lower left), and the ETS mimic (lower right). The Mg²⁺ position in the LTS mimic is similar to the ETS mimic (not shown). The Mg²⁺ ion migrates from the C-site to the position bridging the A9 and scissile phosphates (i.e., directly coordinated with the A9:O2P and C1.1:O2P) in the transition state mimic simulations and in the reactant state simulation with Mg²⁺ initially placed at the C-site position and with C17:O2′ deprotonated, but not in the reactant state simulation with C17:O2′ protonated. The distances shown are distances to Mg²⁺ from A9:O2P, C1.1:O2P, and G10.1:N7.

Figure 2.4

The proposed HHR reaction pathway derived from the free-energy profiles obtained in Section 3.1.

Figure 2.5

(A) Selected 2D surface in 3D free-energy profile simulations, harmonically restrained along the coarse-grained metal ion-binding coordinate at d(Mg, G8:O2′) = 2.5 Å, where z₁ = d(C1.1:O5′,P) − d(P, G8:O2′), z₂ = d(G8:O2′, G8:Ho2′) − d(G8:Ho2′, C1.1:O5′). (B) 2D PMF for Mg²⁺-binding mode in phosphoryl transfer step, where z₄ = d(Mg, O5′)+d(Mg, G8:O2′). (C) 2D PMF for Mg²⁺-binding mode in general acid step, where z₅ = d(Mg,O5′) − d(Mg,G8:O2′). d(x, y) denotes distance between x and y. TS is the acronym of transition state.

Figure 2.6

Schematic view of the coordination sites in the HHR active site. Left: The coordination pattern of Mg²⁺ in the “C-site” coordinated to G10.1:N7 and A9:O2p. Middle: The coordination pattern of Mg²⁺ in the “B-site” bridging A9:O2p and C1.1:O2p of the scissile phosphate. Right: Coordination sites for Na⁺ in the HHR active site found in the RT-Na and dRT-Na simulations. Red numbers next to the coordination sites are the scores used to calculate the coordination index (see text). M1 involves direct binding to A9:O2p and C.1:O2p and indirect binding to G10.1:N7 through a water molecule. M2 involves direct binding to C17:O2′ and C.1:O2p. M3 involves direct binding to C17:O2′ and is positioned toward the outside of the active site.

Figure 2.7

Plot of the C17:O2′ ⋯ P—C1.1:O5′ angle versus C17:O2′—P distance for the approach of the 2′-hydroxyl of residue C17 to the phosphate of residue C1.1 for the reactant state (RT) and the activated state (dRT) simulations. C-Mg indicates that the Mg²⁺ was initially placed at the C-site position, while B-Mg means the Mg²⁺ ion was initially placed in the B-site position. Data obtained from the last 250 ns of the simulations are shown with a frequency of 50 ps and points are colored according to the clustering results and Table 2.6: cluster A (light gray) and cluster B (dark gray). The light gray lines at 3.25 Å and 150 degrees indicate the near in-line attack conformation (NAC) region defined by Torres and Bruice.

Figure 2.8

Two-dimensional radial distribution function of Na⁺ ions in the active site for the activated precursor simulation without Mg²⁺ present in the active site (dRT-Na). The lower panels show results for cluster A that contains population members that are in active in-line conformations, and the upper panels show results for cluster B that are not in-line (see Table 2.6). The axes are the distances (in Å) to different metal ion coordination sites. The light gray lines indicate the regions where Na⁺ ions have distances less than 3.0 Å to both sites indicated by the axes.

Figure 2.9

The 3D density contour maps (white) of Na⁺ ion distributions derived from the RT-Na (upper panels) and dRT-Na simulations (low panels) at different isodensity contour levels (left panels: 0.1; right panels: 1.0). The HHR is shown in dark gray. The figure shows that, although the Na⁺ ions distribute around the RNA phosphate backbone (left panels), the HHR folds to form a local electronegative recruiting pocket that attracts a highly condensed distribution of the Na⁺ ions (left panels) both in the reactant state and the deprotonated activated precursor state (deprotonated C17:O2′) simulations.

Figure 2.10

Schematic representations of the HHR active sites and the two potentially important hydrogen bond networks between C3 and G8 and between G5 and C17. All key structural indexes calculated are also labeled.

Figure 2.11

Schematic representations of the mutants and the hydrogen bonding network of the C3 and G8 positions. k_rel is the experimental cleavage constant relative to the wild type. The relevant references are listed in Table 2.13.

Figure 2.12

Schematic representations of the mutants of the G5 position and the hydrogen bonding network between the G5 and C17 positions. k_rel is the experimental cleavage constant relative to the wild type. The relevant references are listed in Table 2.13.

Figure 2.13

The time series of general acid hydrogen bond indexes, r_HA and θ_HA, as well as the hydrogen bond distance r₃ (see Figs. 2.10 and 2.11), from wild type and G8A activated precursor state simulations. The distances are in Å and the angles are in degrees. These results indicate that the timescale for equilibration and stable fluctuation in some cases exceed 30 ns (shown by vertical dashed line).