Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

Abstract

The relationship between formation of active in-line attack conformations and monovalent (Na(+)) and divalent (Mg(2+)) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg(2+) in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg(2+), in addition to stabilizing active conformations, plays a specific chemical role in catalysis.

Figure 1

Schematic view of the coordination sites in the hammerhead ribozyme active site. Left: The coordination pattern of Mg²⁺ in the “C-site” coordinated to G10.1”N₇ and A9:O_2P. Middle: The coordination pattern of Mg²⁺ in the “B-site” bridging A9:O_2P and C1.1:O_2P of the scissile phosphate. Right: Coordination sites for Na⁺ in the hammerhead ribozyme 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:O_2P and C.1:O_2P and indirect binding to G10.1:N₇ through a water molecule. M₂ involves direct binding to C17:O_2′ and C.1:O_2P . M₃ involves direct binding to C17:O_2′ and is positioned toward the outside of the active site.

Figure 2

Plot of the O_2′–P–O_5′ angle versus O_2′–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 the Mg²⁺ was initially placed at the C-site position while B-Mg means the Mg²⁺ ion was initially placed in an equatorial bridging 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: cluster A (red) and Cluster B (blue). Green lines at 3.25 Å and 150 degrees indicate the near in-line attack conformation (NAC) region defined by Torres and Bruice.

Figure 3

Plot of the in-line attack angle (O_2′–P–O_5′) in degrees and the coordination index of Na⁺ ions for the RT-Na (top) and dRT-Na (bottom) simulations. The coordination index is defined as follows: when a Na⁺ ion has a distance less than a 3.0-Å cutoff value to a ligand, it is defined as bound to that ligand for indexing purposes. When an ion is bound, the scores for the four possible coordination sites are 1 for G8:O_2′ , 2 for A9:O_2P , 4 for C1.1:O_2P , and 8 for C17:O_2′. The coordination index of a single Na⁺ ion is the sum of all scores from its bound sites. Individual Na⁺ ions are tracked using different colors (red, green, blue and yellow). Data obtained from the last 250 ns are shown in steps of 500 ps.

Figure 4

Two dimensional radial distribution function of Na⁺ ions in the active site for the activated precursor simulation without without Mg²⁺ present in the active site (dRT-Na). The lower panels shows 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). The axes are the distances (in Å) to different metal coordination sites. The green lines indicate the regions where Na⁺ ions have distances less than 3.0 Å to both sites indicated by the axes.

Figure 5

The 3D density contour maps (yellow) 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 hammerhead ribozyme is shown in blue with the active site highlighted red. The figure shows that, although the Na⁺ ions distribute around the RNA phosphate backbone (left panels), the hammerhead ribozyme 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:O_2′) simulations.