Figure 2. Computational investigation of catalytic strategies adopted by the VS ribozyme.

Panels (a) and (b) summarize results from MD simulations based on the existing crystal structures as is. Panel (a) illustrates the persistence of active site interactions during the various MD simulations. The three columns correspond to simulations departing from the 4R4P, 4R4V, and 5V3I crystal structures, while the rows correspond to interactions related to different catalytic strategies (α-δ). The first row refers to in-line fitness, which is dictated by the τ (O2’-P-O5’) angle ≥ 140° and the O2’-P distance ≤ 3.5 Å. Hydrogen bond and metal-ion interaction distance thresholds (β-δ) were chosen to ≤ 2 Å and ≤ 2.5 Å, respectively. Panel (b) is 2D scatter plot of in-line attack angle τ and O2’-P distance (r), and include data from all MD simulations. Data points are classified into 4 categories – configurations that exhibit both α and γ catalysis (magenta), only α catalysis (blue), only γ catalysis (red), and configurations that do not exhibit either α or γ catalysis (non-active, gray). Datapoints corresponding to the different categories are indicated by arrows and the black rectangle outlines the region that exhibits α catalysis. Panel (c) illustrates Na⁺ ion charge isodensity plots in the active site region calculated from MD simulations. To construct these isodensity plots, a 3D cubic grid having an edge length of 12 Å and spacing of 1 Å was centered on the scissile phosphate phosphorus atom. For a given MD trajectory, the total charge per grid cell was determined by summing over the charges of all ions in that particular cell. The blue solid surface and the mesh represent the top 30% and top 70% of the charge density of the entire grid, respectively. Panel (d) illustrates Mg²⁺ ion binding site predicted by 3D-RISM calculations. The magenta surface represents the top 10% of Mg²⁺ ion isodensity. Panel (e) depicts the Mg²⁺ bound active site model obtained from MD simulations. In this model, the Mg²⁺ ion forms inner sphere contacts with the A621 pro-S_P NPO, A622 pro-R_P NPO and G623:N7, and interacts with G638:O6 via a water molecule. Panels (f) and (g) are analogous to panels (a) and (b), respectively, and summarize results from MD simulations based on the crystal structures but with an added Mg²⁺ ion bound at the pro-S_P NPO of the scissile phosphate.