Metal ions are essential for many cellular processes such as DNA replication, DNA repair, and transcription. DNA replication, is catalyzed by a family of enzymes called DNA polymerases. The classical two metal ion mechanism has previously been proposed for DNA polymerases. The catalytic Mg2+ reduces the pKa of the growing strand’s 3’-OH group to prepare for nucleophilic attack; the nucleotide bound magnesium ion helps to stabilize ground state binding. The ion positions have been determined using crystallography for many DNA polymerases, which only provides a static picture that cannot explain the dynamic mechanism of Mg2+ ion incorporation: it’s coupling with the incoming nucleotide and its effect on the electrostatic environment at the active site. We studied Mg2+ ion coordination in HIV-1 reverse transcriptase (HIVRT) upon nucleotide incorporation using Molecular Dynamics (MD) simulation. Brute force MD simulations of an ensemble of Mg2+ ion association events enabled a detailed study of the binding mechanism of metal ions. Using Milestoning theory we computed the rate of Mg2+ ion association to the active site. Nucleotide binding in different ion binding states was studied using Potential of Mean Force computations. Strong coupling between magnesium localization and substrate binding was observed in equilibrium simulations, enabling us to study the electrostatic environment for both open and closed states before and after the chemical reaction. Based on these simulations, we propose that metal ions have further roles in maintaining the mechanism of action of HIVRT, particularly on its fidelity. Integrating kinetics and thermodynamics of magnesium ion association obtained from these simulations allowed us to resolve the order of events governing the incorporation of the incoming nucleotide in the presence of metal ions. Our results show that magnesium ions play an active role in regulating the enzyme function, a role that goes beyond catalysis. Our data agrees well with recent stopped flow kinetic experiments, and reveal new insights into the metal ion mechanism of DNA polymerases by providing an atomic-level physics-based description of this process.

Computational study of magnesium ion coordination in the mechanism of HIVRT Polymerase

255th National Meeting (2018)

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