Molecular dynamics study displays near in-line attack conformations in the hammerhead ribozyme self-cleavage reaction

Abstract

We have performed molecular dynamics (MD) calculations by using one of the recently solved crystal structures of a hammerhead ribozyme. By rotating the ␣, ␤, ␥, ␦, , and torsion angles of the phosphate linkage of residue 17, the nucleobase at the cleavage site was slightly rotated out of the active site toward the solution. Unconstrained MD simulations exceeding 1 ns were performed on this starting structure solvated in water with explicit counter ions and two Mg 2؉ ions at the active site. Our results reveal that near attack conformations consistently were formed in the simulation. These near attack conformations are characterized by assumption of the 2-hydroxyl to a near in-line position for attack on the -O-(PO 2 ؊ )-O-phosphorous. Also during the time course of the MD study, one Mg 2؉ moved immediately to associate with a pro-R phosphate oxygen in the conserved core region, and the second Mg 2؉ remained associated with the pro-R oxygen on the phosphate linkage undergoing hydrolysis. These results are in accord with a one-metal ion mechanism of catalysis and give insight into the possible roles of many of the conserved residues in the ribozyme.The hammerhead ribozyme is one of a small class of selfcleaving RNAs that catalyzes the hydrolysis of phosphate esters (1-4). This ribozyme consists of a conserved core of 15 nucleotides required for full activity formed by three base pairing stem regions (5). The secondary structure of the hammerhead ribozyme studied here is shown in Chart 1 by using the standard nomenclature (6), with the conserved bases shown in outlined letters and the cleavage site indicated by the arrow. Note that the hammerhead ribozyme structure of Chart 1 exists as two separate strands with functions of enzyme (E) and substrate (S) such that it cleaves in trans (4, 7).The hammerhead ribozyme catalyzes the chemically well studied RNA hydrolysis mechanism (8), which involves in-line nucleophilic attack of the 2Ј-hydroxyl on phosphate phosphorous at the 3Ј-position with elimination of 5Ј-hydroxyl and formation of a 2Ј,3Ј-cyclic phosphate ester with inversion of configuration at the phosphorus (Scheme 1 refs. 9-11).Hammerhead ribozyme solvolysis is first order in HO Ϫ and requires one or more divalent metal ions (10, 12, 13). Thus, two kinetically equivalent mechanisms may be considered. The first is that metal ligated hydroxide acts as a general base to initiate the reaction by forming the 2Ј-O Ϫ nucleophile at residue 17. A second possibility is specific base catalysis of 2Ј-O Ϫ formation by lyate HO Ϫ and participation of the metal ion elsewhere. The metal ion may participate in various ways. It has been shown that a metal ion must ligate to the pro-R oxygen of the scissile phosphate group undergoing reaction (14,15). By doing so, the negative charge on the phosphate is neutralized and facilitates nucleophilic attack on the phosphorus atom. This divalent metal ion or possibly an additional one can carry out the required stabilization of the developing negative charge on the 5Ј-leaving gr...

Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

“…The light gray lines at 3.25 Å and 150 degrees indicate the near in-line attack conformation (NAC) region defined by Torres and Bruice. 135 …”

Quantum Chemical Studies of Intermediates and Reaction Pathways in Selected Enzymes and Catalytic Synthetic Systems

“…123,124 The energy associated with the highest lying transition state, TS2, shows that one metal ion is sufficient to catalyze the self-cleavage of the hammerhead ribozyme. The possibility of two metal ions participating in the cleavage reaction, however, cannot be eliminated.…”

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