The Mechanism of Phosphodiester Hydrolysis: Near In-line Attack Conformations in the Hammerhead Ribozyme

Abstract

The hammerhead ribozyme is a small RNA molecule capable of self-cleavage at a specific site in the phosphodiester backbone. The mechanism of hydrolysis involves in-line nucleophilic attack by the 2‘-hydroxyl of residue 17 on the adjoining phosphorus of residue 1.1, resulting in the formation of a 2‘,3‘-cyclic phosphate ester on residue 17 (C17) and elimination of the 5‘-hydroxyl group of residue 1.1 (A1.1). Unconstrained molecular dynamics (MD) simulations on the recently solved crystallographic unmodified hammerhead ribozyme structure were performed in solution using two crystallographic Mg2+ ions. The simulations indicate that near in-line attack conformations (NACs), in which the distance of the 2‘-oxygen of C17 to the phosphorus of A1.1 is ≤3.25 Å and the C17 2‘O−A1.1 P−A1.1 O5‘ angle of displacement is ≥150°, form approximately 18% of the simulation time. The motions leading to these catalytically competent conformations are discussed. Stems I and II of the hammerhead ribozyme structure, released from the pseudo-continuous helix and other crystallographic constraints, move toward each other. This, along with the Mg2+ ion bound at the pro-R phosphate oxygen of residue 1.1, prompts torsional rotations in the phosphodiester backbone primarily near the active site. These rotations lead to the unstacking of residues C17 and G5 from A6. Residue G5 then interacts with other conserved residues in the structure and does not stack with A6 again. Following spontaneous backbone conformational rearrangements, a ribose sugar pucker flip from C3‘-endo to C2‘-endo occurs in the nucleotide containing the 2‘-hydroxyl nucleophile. The base of residue C17 then restacks with the base of residue A6, and NACs occur shortly thereafter. During the simulations, one Mg2+ ion remains coordinated to the pro-R phosphate oxygen of the C17 nucleotide, while the other Mg2+ ion serves a structural role and does not participate in the transesterification reaction. The formation of NACs is spontaneous during the simulation and in a replicate simulation.