Solvent structure and hammerhead ribozyme catalysis

Abstract

Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.

Figure 1. Wall-Eyed Stereo Representation of the Hammerhead Ribozyme

(A) Stereo view of the active site with mechanistically relevant contacts indicated. G12 adopts a position in the hammerhead ribozyme active site consistent with the role of a general base catalyst in the cleavage reaction (assuming the N1 of G12 is abstracted), and the 2′-OH of G8 is positioned in such a way that it may participate in general acid catalysis by donating a proton to the 5′ oxygen leaving group of C1.1 as negative charge accumulates during bond scission. A Mn²⁺ ion (magenta) is coordinated by the pro-R oxygen of the A9 phosphate and the N7 of G10.1, as well as four water molecules (red spheres). These two purines also serve to poise G12 for general base catalysis. An ordered water (W, in red) or other solvent molecule is also bound at the cleavage site. It is within hydrogen-bonding distance to the N1 of G12 and the 2′-OH of G8 (yellow, dotted lines), the two functional groups thought to be involved in the proton transfers required for initiating and terminating catalytic cleavage, respectively. The orange, dotted lines indicate possible routes of proton abstraction by G12. Other hydrogen bonds are shown as white, dotted lines, and the covalent bond that will form upon activation of the nucleophile is shown as a light-blue, dotted line. The 2′ -CH₃ that prevents cleavage in the crystal has been omitted from the figure for clarity. (B) Overview of water and Mn²⁺-ion-binding sites in the hammerhead ribozyme crystal structure. Water (small, orange spheres) and Mn²⁺ (large, white spheres) sites on the full-length hammerhead ribozyme are indicated. The Mn²⁺ sites were identified by using an F(+) − F(−) anomalous difference Fourier as described in the text. The color scheme corresponds to that used to describe the full-length hammerhead ribozyme structure (Martick and Scott, 2006). (C) A close-up of Mn²⁺ site C near the cleavage site of the hammerhead ribozyme. The F(+) − F(−) anomalous difference Fourier permits unambiguous identification of high-occupancy Mn²⁺-ion-binding sites. Shown as a magenta mesh corresponding to an anomalous difference Fourier peak contoured at 10σ, and displayed by using COOT (Emsley and Cowtan, 2004), the C site Mn²⁺ ion coordinates the N7 of G10.1, the pro-R oxygen of the A9 phosphate, and four water molecules. Oxygen atoms, including those of the water molecules, are shown in red, carbon atoms are shown in white, and nitrogen atoms are shown in blue. (D) Stereo view of the 2F_o −F_c electron density map in the vicinity of the C site Mn²⁺ ion. The final refined σA-weighted 2F_o − F_c electron density map contoured at 1.25 rmsd is shown as a blue mesh superimposed on the refined structure at 2.0 Å resolution. The atomic coloring scheme, as well as that of the contacts, corresponds to (A).

Figure 2. A Schematic Diagram Depicting Three Possible Catalytic Reaction Mechanisms

(A) Scenario 1, wherein only indirect metal ion participation takes place. This is the most conservative hypothesis in that it is consistent with the present crystal structural analysis as well as the previous observation that the hammerhead ribozyme does not strictly require divalent metal ions for efficient catalysis (Murray et al., 1998; Scott, 1999). (B) Scenario 2, a possible single-metal mechanism. (C) Scenario 3, a possible double-metal mechanism. Each scenario is described in detail in the text.