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

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 (C 17 ) and elimination of the 5‘-hydroxyl group of residue 1.1 (A 1.1 ). Unconstrained molecular dynamics (MD) simulations on the recently solved crystallographic unmodified hammerhead ribozyme structure were performed in solution using two crystallographic Mg 2+ ions. The simulations indicate that near in-line attack conformations (NACs), in which the distance of the 2‘-oxygen of C 17 to the phosphorus of A 1.1 is ≤3.25 Å and the C 17 2‘O−A 1.1 P−A 1.1 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 Mg 2+ 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 C 17 and G 5 from A 6 . Residue G 5 then interacts with other conserved residues in the structure and does not stack with A 6 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 C 17 then restacks with the base of residue A 6, and NACs occur shortly thereafter. During the simulations, one Mg 2+ ion remains coordinated to the pro - R phosphate oxygen of the C 17 nucleotide, while the other Mg 2+ 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.