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 Mg2+ 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 less than or equal to 3.25 Angstrom and the C-17 2'O-A(1.1) P-A(1.1) O5' angle of displacement is greater than or equal to 150 degrees, 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: 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 C17 then restacks with the base of residue A(6), and NACs occur shortly thereafter. During the simulations, one Mg2+ ion remains coordinated to the pro-R phosphate oxygen of the C-17 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.