Active participation of Mg ion in the reaction coordinate of RNA self-cleavage catalyzed by the hammerhead ribozyme.

We report results from combined quantum mechanical/molecular mechanical (QM/MM) free energy simulations to explore metal-assisted phosphoryl transfer and general acid catalysis in the extended hammerhead ribozyme. The mechanisms considered here assume that the 2′OH group of C17 has already been activated (i.e., is deprotonated) and acts as a nucleophile to go on an inline attack to the adjacent scissile phosphate, passing through a pentavalent phosphorane intermediate/transition state, followed by acid-catalyzed departure of the O5′ leaving group of C1.1. A series of six two-dimensional potential of mean force profiles are reported in this study, requiring an aggregate of over 100 ns of QM/MM simulation. The simulations employ the AM1/d-PhoT semiempirical quantum model and linear-scaling QM/MM-Ewald method and explore mechanistic pathways for the selfcleavage. Results support the plausibility of a cleavage mechanism where phosphoryl transfer and general acid catalysis are stepwise, and where the catalytic divalent metal ion plays an active role in the chemical steps of catalysis. Small self-cleaving ribozymes such as the hammerhead ribozyme (HHR) have been instrumental as model systems for RNA catalysis. Recently, an extended HHR structure was determined by X-ray crystallography at 2.0 Å resolution, in which a divalent metal ion was observed near the active site. Subsequent computer simulations lend credence to the possibility that this metal ion may play an active role in catalysis, although free energy profiles to elucidate specific pathways have not yet been reported. We report results from combined quantum mechanical/ molecular mechanical (QM/MM) free energy simulations to explore metal-assisted phosphoryl transfer and general acid catalysis in the extended HHR. The mechanisms considered here assume that the 2′OH group of C17 has already been activated (i.e., is deprotonated) and acts as a nucleophile to go on an in-line attack to the adjacent scissile phosphate, passing through a pentavalent phosphorane intermediate/transition state, followed by acid-catalyzed departure of the O5′ leaving group of C1.1. The general acid is assumed to be the 2′OH group of G8. A series of six 2D potential of mean force (PMF) profiles is herein reported, requiring an aggregate of over 100 ns of QM/ * Corresponding author e-mail: [email protected]. † University of Minnesota. ‡ Rutgers University. Table 1. Relative Free Energies and Internuclear Distances at Various States of RNA Self-Cleavage Catalysis in Hammerhead Ribozymes react TS1 int TS2 prod Nu-P 3.50(04) 1.76(05) 1.66(03) 1.67(03) 1.68(03) P-Lea 1.65(03) 2.11(05) 4.51(04) 4.24(48) 3.63(23) gA-H 0.96(00) 0.96(00) 0.96(00) 1.78(04) 3.75(04) H-Lea 2.57(51) 4.07(47) 4.13(73) 1.04(03) 1.00(03) Mg-Lea 3.99(18) 3.61(17) 2.02(05) 2.83(86) 4.48(05) Mg-gA 4.56(18) 4.03(18) 4.33(06) 3.38(86) 2.03(05) ∆G 0.0(4) 24.4(6) -6.7(3) 13.7(7) -13.6(9) a Free energies (∆G) are in kcal/mol, which were extracted from 1D PMF profiles along the minimum free-energy path through the 2D profiles. Average distances (X-Y) are in Å. Standard deviations are listed in parentheses divided by the decimal precision of the average values. The abbreviations “react”, “TS”, “int”, and “prod” signify reactant, transition, intermediate, and product states, respectively, and for the distance metrics, “Nu”, “Lea”, “gA”, and “H” refer to the O2′ nucleophile, O5′ leaving group, general acid residues G8:O2′, and H2′, respectively. Figure 1. Schematic diagram for relative free-energy barriers and the corresponding reaction rate constants. J. Chem. Theory Comput. 2011, 7, 1–3 1 10.1021/ct100467t  2011 American Chemical Society Published on Web 12/16/2010 MM simulation. Simulations were based on the extended HHR solvent structure (PDB: 2OEU) solvated by over 10 000 water molecules. Active site residues (G8, A9, C1.1, C17, and an Mg ion with coordinated water molecules shown in Figure 3) were treated quantum mechanically (81 atoms total) using the AM1/d-PhoT quantum model with the AM1/d model for Mg. We used the all-atom AMBER parmbsc0 force field, to describe the HHR outside of the active site, along with the TIP4P-Ewald water model and the consistent set of monovalent ion parameters. Multidimensional PMF profiles were generated along reaction coordinates corresponding to phosphoryl transfer, proton transfer from the general acid to the leaving group, and the divalent metal ion binding mode. Complete details are given in the Supporting Information. Phosphoryl transfer and general acid steps are stepwise, and sensitiVe to the Mg binding mode. Our initial attempts to study the chemical steps of the HHR reaction from 2D PMF profiles using phosphoryl transfer and proton transfer reaction coordinates, but not considering a reaction coordinate associated with Mg ion binding mode, led to free energy barriers that were unexpectedly high (∼37 kcal/mol) compared to an estimated barrier of ∼20 kcal/mol derived from the experimental rate of one turnover per minute in HHR catalysis. We extended the calculations so as to include a 3D-PMF profile with a coursegrained reaction coordinate associated with the Mg binding mode and confirmed the sensitivity of the barriers to the Mg ion position along the reaction coordinate. A common feature of the reaction mechanism derived from the 3D profile was that the phosphoryl transfer and general acid steps were stepwise (e.g., Figure 2a), allowing these steps to be decoupled. Both the phosphoryl transfer and general acid steps of the reaction were coupled with the Mg binding mode, and hence separate 2D profiles were generated for each step with a reaction coordinate corresponding to the Mg binding mode as a second dimension. Table 1 summarizes key average geometrical parameters, and free energy values for stationary points along the reaction. The corresponding reaction rate constants are depicted in Figure 1. Phosphoryl transfer is rate-limiting and facilitated by electrostatic stabilization by Mg. The phosphoryl transfer step is rate-controlling, having a free energy barrier of approximately 24.4 kcal/mol. The position of the Mg ion follows the negative charge along the phosphoryl transfer reaction coordinate in order to provide electrostatic stabilization. The change in the Mg position is continuous and monotonic throughout the phosphoryl transfer step (Figure 2b) and is most pronounced in the initial and final stages when the nucleophile and leaving group have the greatest negative charge. The transition state is late (Figure 3), having a P-O5′ distance of 2.11 Å. As the P-O5′ bond breaks, the Mg ion forms an innersphere coordination, leading to a Mg-bound O5′ alkoxide intermediate. General acid catalysis is concerted with changes in the Mg binding mode. The general acid step considered here assumes that the 2′OH of G8 acts as a general acid catalyst to transfer a proton to the O5′ leaving group. An examination of Figure 2c indicates that proton transfer occurs after formation of the Mg-coordinated cleaved intermediate and is concerted with changes in Mg binding mode. Unlike the phosphoryl transfer step, participation of the Mg along the reaction coordinate is most pronounced not at the end points of the step but near the midpoint where the proton transfer occurs. The free energy barrier for the transition state of the general acid step (Figure 3) is 13.7 kcal/mol with respect to the activated precursor state. The intermediate is only 6.7 kcal/mol lower in Figure 2. (A) Selected 2D surface, harmonically restrained along the course-grained metal ion binding coordinate at d (Mg, G8:O2′) ) 2.5 Å, where z1 ) d(O5′,P) d(P,O2′), z2 ) d(G8:O2′,H) d(H,O5′). (B) 2D PMF for the Mg binding mode in the phosphoryl transfer step, where z4 ) d(Mg, O5′) + d(Mg, G8:O2′). (C) 2D PMF for the Mg binding mode in the 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 3. Snapshots of the active site at the transition states for phosphoryl transfer (left) and general acid catalysis (right) with average distances labeled. 2 J. Chem. Theory Comput., Vol. 7, No. 1, 2011