Investigation of the p Ka of the Nucleophilic O2' of the Hairpin Ribozyme

Abstract

Small ribozymes cleave their RNA phosphodiester backbone by catalyzing a transphosphorylation reaction wherein a specific O2' functions as the nucleophile. While deprotonation of this alcohol through its acidification would increase its nucleophilicity, little is known about the pK_a of this O2' in small ribozymes, in part because high pK_a's are not readily accessible experimentally. Herein, we turn to molecular dynamics to calculate the pK_a of the nucleophilic O2' in the hairpin ribozyme and to study interactions within the active site that may impact its value. We estimate the pK_a of the nucleophilic O2' in the wild-type hairpin ribozyme to be 18.5 ± 0.8, which is higher than the reference compound, and identify a correlation between proper positioning of the O2' for nucleophilic attack and elevation of its pK_a. We find that monovalent ions may play a role in depression of the O2' pK_a, while the exocyclic amine appears to be important for organizing the ribozyme active site. Overall, this study suggests that the pK_a of the O2' is raised in the ground state and lowers during the course of the reaction owing to positioning and metal ion interactions.

Figure 1.

The active site of the hairpin ribozyme. An MD configuration of the entire ribozyme is shown to the left with the active site colored red and magnified as a drawing on the right. In the drawing, the electron pushing arrows illustrate how transphosphorylation occurs between A-1 and G1. G8 and A38 are catalytically important residues that hydrogen bond with the scissile phosphate. Hydrogen bonds observed in the WT hairpin ribozyme simulations in this study are drawn. The G8(N2)-to-G1(pro-S_P NPO) hydrogen bond is colored blue and is absent in simulations of the G8I variant.

Figure 2.

Na⁺ ion binding to A-1(O2′) in ApG and the hairpin ribozyme. A) Na⁺ ion isodensity plots for the O2′ protonated ribozyme. The transparent purple corresponds to the top 70% of Na⁺ ion density within a 10x10x10 ų region centered near the O2′. Residues A-1 and G8 are labeled, an arrow points to the O2′, and the carbons of A-1 and G1 are cyan while all others are grey. B) Running coordination number of Na⁺ ions to the protonated O2′ for ApG (black) and the ribozyme (blue). C) Same as panel A except for the deprotonated O2′, and the opaque and transparent purple correspond to the top 30% and 85%, respectively, of Na⁺ ion density. D) Same as panel B except for the deprotonated O2′. Also note the differences in the y-axis values between panels B and D.

Figure 3.

Solvent accessible surface areas and hydration of A-1(O2′) in ApG and the hairpin ribozyme. A) Distributions of the SASA of the protonated O2′ in ApG (black) and the ribozyme (blue). The area under each distribution is normalized to one. B) Running coordination number of water hydrogen atoms to the protonated O2′ in ApG (black) and the ribozyme (blue). C) and D) Same as panels A and B except for the deprotonated O2′.

Figure 4.

Hydrogen bonding of the protonated O2′ to the NPOs in ApG and the hairpin ribozyme. MD configurations of the ApG simulations show an example of a hydrogen bond being donated from the O2′ (designated by the arrow) to A) the pro-S_P NPO or B) the pro-R_P NPO. C) and D) Heatmaps of hydrogen bonding distances and angles for ApG where the O2′ is the donor and the acceptors are either the pro-S_P NPO (panel C) or the pro-R_P NPO (panel D). E) and F) Same as panels C and D, respectively, except for the ribozyme. In all four heatmaps, the same number of data points are binned, and colors correspond with the color bar at the right. The red box in each heatmap designates good hydrogen bonding geometry with distances less than or equal to 3.2 Å and angles greater than or equal to 135°, and the percentages depicted represent the fraction of points that meet the hydrogen bonding criteria.

Figure 5.

Distances between the deprotonated O2′ and NPOs in ApG and the hairpin ribozyme. A) and B) O2′ to pro-S_P NPO (panel A) and pro-R_P NPO (panel B) distances throughout the 500 ns trajectory for ApG (black). C) and D) Same as panels A and B, respectively, except for the hairpin ribozyme (blue).

Figure 6.

In-line fitness of ApG and the hairpin ribozyme in the O2′ protonated state. A) and B) Heatmaps of in-line fitness distances and angles for ApG (panel A) and the ribozyme (panel B). Points that fall within the green box are considered fit for nucleophilic attack, and the percentages depicted represent the fraction of points that meet the in-line fitness cutoffs. In both heatmaps, the same number of data points are binned, and colors correspond with the color bar in panel B. C) Drawing of the phosphate linkage showing the O2′ positioned for in-line nucleophilic attack, with the O2′, phosphorus, and O5′ circled in green. D) and E) Isodensity plots of the O2′ relative to the phosphate of G1 in ApG (panel D) and the ribozyme (panel E). The top 90% of O2′ density is shown in red and overlayed on an MD configuration for reference. Density that falls within the cone is considered fit for in-line nucleophilic attack. Alternative perspectives are shown in Figure S3.

Figure 7.

Distances of the protonated O2′ to the NPOs of ApG. A) Heatmap of the O2′ to pro-R_P and pro-S_P NPO distances for the entire trajectory of ApG. Colors correspond to the color bar at the right, and the diagonal line shows where the O2′ to pro-R_P and pro-S_P NPO distances are equal. B) Same as panel A except the analysis is performed for only the fraction of the trajectory where the O2′ is positioned for in-line nucleophilic attack. Note that the counts are lower due to this being a subpopulation of the whole trajectory.

Figure 8.

Conformational changes in the G8I variant hairpin ribozyme simulations. The values of in-line fitness (grey), the epsilon dihedral (black) and the sugar pucker (blue) of A-1 throughout the 500 ns trajectory are shown when the O2′ is A) protonated and B) deprotonated. The red vertical lines separate regions with different conformations and are drawn at 345 ns in panel A and at 29.1 ns and 426.6 ns in panel B. For comparison of the G8I variant to WT, the dashed red horizontal lines mark the average in-line fitness from the WT simulations, which are 0.45 for the protonated state (panel A) and 0.43 for the deprotonated state (panel B). C) MD configurations from the O2′ deprotonated trajectory exemplify the different conformations observed in panels A and B. Atoms that form the epsilon dihedral plotted in panels A and B are identified by the black traces. Residues A-1 and I8 are labeled. Red numbers depicted in each configuration correspond to the regions in the plots, separated by red vertical lines and also labeled with red numbers.