Ab initio and QM/MM Calculations on a Prototype of Catalytic RNA

Fabrice Leclerc & Martin Karplus
UniversitŽ Louis Pasteur, Institut Le Bel
Laboratoire de Chimie Biophysique
4, rue Blaise Pascal, Strasbourg 67000, France

Introduction

The hammerhead ribozyme is one of the smallest catalytic RNAs known as catalyst for the site-specific cleavage of a phosphodiester linkage. Because of its small size, it has been extensively studied as a prototype for catalytic RNAs. The recent determination by X-ray crystallography of several conformational intermediates of this biologically active ribozyme (Fig. 1) provides a framework from which a detailed molecular mechanism of catalysis can be modeled. So far, no theoretical study has been done on catalytic RNAs. Thus by this study we intend to propose the first detailed mechanism for a ribozyme and investigate the role of the divalent metal ions and the active site surrounding in the catalysis.

Figure 1: X-ray structure of the hammerhead ribozyme. The phosphodiester backbo ne is represented by a white ribbon. The active site is included in a yellow circle where the atoms are colored according a standard scheme (carbon: green, oxygen: red, phosphorus: purple). T he bond cleaved during the phosphate ester hydrolysis (in red) is indicated by a purple arrow. Mg²⁺ cations are cofactors in this reaction, they are represented by a van der Waals spheres (orange).

The reaction proceeds via an in-line attack on the phosphodiester bond: the activated 2'O^-species generated by deprotonation of the 2'OH of the attacking nucleotide acts as nucleophile, the leaving group corresponding to a 5'OH species. The reaction proceeds via an in-line attack of the activated 2'O^- on the phosphodiester bond leading to an inversion of configuration in the stereochemistry of the phosphate group (Fig. 2). Two mechanisms have been proposed for the reaction; they essentially differ by the way the 2'O^- species is generated: in the di-anionic mechanism, the proton is taken from the solution while in the mono-anionic mechanism, it is an internal proton transfer. Thio analogs of the natural RNA substrate have been used to infer the ligand sites to the metal ions used as cofactors in the reaction. However, the exact metal coordinations are not known and there is no strong evidence for inner or outer sphere coordinations.

Figure 2: Detailed view of the active site based on a two-metal-ion mechanism. The arrow (purple) represents the nucleophilic attack of the O2' oxygen on the phosphate group. The attack that proceeds via an in-line mechanism (alignment of O2', P and O5') leads to the formation of the O2'-P bond and the breaking of the P-O5' bond.

Preliminary Results

Three different thio analogs of the natural RNA substrate have been studied experimentally: in the first one (pro-RpS), the pro-R non-bridging oxygen of the phosphate group is replaced by sulfur; in the second one (pro-RpS, pro-SpS) both the pro-R and pro-S non-bridging oxygen atoms are replaced by sulfur; in the last one the bridging O5' oxygen corresponding to the leaving group is replaced by sulfur. The pro-RpS analog is cleaved at a lower rate but the catalytic activity is partially restored by substituting the normal metal cofactors (rescue effect), Mg²⁺, by more thiophilic metal ions such as Mn²⁺ or Cd²⁺ [Scott2]. On the other hand, the dithioate analog (pro-RpS, pro-SpS) is still cleaved in presence of Mg²⁺ though at a lower rate.

The studies have been carried out on a small model representing a minimum active site of the hammerhead ribozyme. The RNA model used in the study includes a ribose moiety, a methyl-phosphate group and two solvated metal ions (Mg²⁺). Different metal configurations involving inner or outer-sphere coordinations have been explored. We have identified two configurations favorable to the in-line attack: the coordinations of the two metal cations were inferred from experimental data on the ``rescue effect'', and from ab initio calculations at the Hartree-Fock level (HF/3-21+G*).

Research Proposal

The two configurations favorable to the in-line attack will be used as initial geometry to follow the reaction path. In the first configuration, the two metal ions have inner-sphere coordinations with the O2' and the pro-R oxygen for the first metal, and with the O5' and pro-R oxygen for the second metal. We will study the hydrolysis of the mono and dithioate analogs but only the intermediates on the reaction path will be calculated. To reproduce the experimental conditions of the hydrolysis of the pro-RpS analog in our calculations, only one of the two Mg²⁺ will be replaced by Cd²⁺: two cases will be considered since the thiophilic metal Cd²⁺ can be coordinated to a sulfur at both metal sites. In the second configuration, only the metal stabilizing the leaving group at the O5' site has a inner-sphere coordination. Thus, the metal at this position will be replaced by Cd²⁺ for the calculations on the pro-RpS analog. The calculations for the hydrolysis of the dithioate analog will be carried out with two Mg²⁺ ions, as measured experimentally (Derrick & Uhlenbeck, personal communication).

Methods

The reaction path will be followed by QM/MM calculations where the RNA model and the two metal ions are treated by quantum mechanics and the water molecules coordinated to the metal ions by molecular mechanics. The CHARMM/GAMESS program (the GAMESS ab initio package interfaced to the CHARMM program) is used to model the reaction path from the reactants (initial geometries) to the products by imposing distance constraints on the O2'-P and P-O5' bonds corresponding to an antisymmetric stretch.

Figure 2: Detailed view of the active site based on a two-metal-ion mechanism. The arrow (purple) represents the nucleophilic attack of the O2' oxygen on the phosphate group. The attack which proceeds via an in-line mechanism (alignment of O2', P and O5') leads to the formation of the O2'-P bond and the breaking of the P-O5' bond.