Magnesium-Dependent Active-Site Conformational Selection in the Diels−Alderase Ribozyme

Abstract

The Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme, accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition, a reaction with broad application in organic chemistry. Here, the Diels-Alderase ribozyme is examined via molecular dynamics (MD) simulations in both crystalline and aqueous solution environments. The simulations indicate that the catalytic pocket is highly dynamic. At low Mg(2+) ion concentrations, inactive states with the catalytic pocket closed dominate. Stabilization of the enzymatically active, open state of the catalytic pocket requires a high concentration of Mg(2+) ions (e.g., 54 mM), with cations binding to specific phosphate sites on the backbone of the residues bridging the opposite strands of the pocket. The free energy profile for pocket opening at high Mg(2+) cation concentration exhibits a double minimum, with a barrier to opening of approximately 5.5 kJ/mol and the closed state approximately 3 kJ/mol lower than the open state. Selection of the open state on substrate binding leads to the catalytic activity of the ribozyme. The simulation results explain structurally the experimental observation that full catalytic activity depends on the Mg(2+) ion concentration.

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

“…The pro-R P phosphate oxygen of nucleotide U20 is located 4.0–4.5 Å away from both Mg1 and Mg2, which is a typical distance for outer sphere interactions and may explain the weaker interference. The dimeric metal ion site was also supported by EPR-spectroscopy that identified—by a distinct coupling pattern—two metal ions in close proximity (∼7 Å distance) (14), and by MD simulations (15). …”

“…Intriguingly, MD simulations recently established the existence of Diels–Alderase ribozyme conformations with a closed pocket (15). These species still contain the global nested pseudoknot fold, but the register of the upper pseudoknot minihelix is shifted (G2:C11 and G1:A12 instead of G2:C10 and G1:C11), the stabilizing interaction with U17 is missing, and nucleotide G2 stacks directly on A3, thereby closing the catalytic pocket.…”

“…The concentration of divalent metal ions was established as a major determinant of folding and dynamics (12–14). Recent molecular dynamics (MD) simulations revealed the existence of conformations with similar overall architecture but either closed or open catalytic pocket, resulting in either inactive or active catalysts (15). …”

Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis

“…[26][27][28] Single-molecule FRET measurements and MD simulations on the apo-DAse revealed a dynamic ribozyme structure where the distribution of states was dependent on the Mg 2+ concentration. 5,29 In those MD simulations, the catalytic pocket was found to have two possible conformations, an open and a closed state. In the open state, up to 23 water molecules were observed inside the catalytic pocket, while in the closed state the catalytic pocket had collapsed and no water molecules could be accommodated.…”

“…In the open state, up to 23 water molecules were observed inside the catalytic pocket, while in the closed state the catalytic pocket had collapsed and no water molecules could be accommodated. 29 Therefore, binding of NPM to the open state DAse would result a priori in a loss of bound water from the catalytical pocket (Fig. 4B).…”