Understanding the R882H mutation effects of DNA methyltransferase DNMT3A: a combination of molecular dynamics simulations and QM/MM calculations

Abstract

DNA (cytosine-5)-methyltransferase 3A (DNMT3A), a key enzyme for de novo epigenetic methylation in human beings, was reported to undergo an R882H mutation in approximately 25% of M4/M5 subtype acute myeloid leukemia (AML) patients. In this work, a combination of classical molecular dynamics (MD) simulations and QM/MM calculation methods was utilized to reveal the molecular mechanism behind the activity attenuation caused by R882H mutation. We found that R882H mutation induces a "folded" conformation in the methyl donor S-adenosylmethionine (SAM) through different types of hydrogen bond formation at the terminal carbonyl oxygen atom and the hydroxyl O3' atom of the ribose ring on SAM, with Arg891 as a mediator. Energetically, both the pre-reaction state (PRS) and transition state (TS) were stabilized in the R882H mutant. However, the energy barrier of the rate-determining step from the PRS to the TS was calculated to be roughly 1.0 kcal mol^-1 larger in the R882H mutant than the WT. Also, a dynamic transformation occurred along the helix where R882H was located, tending to manifest in a quasi-"Newton's cradle" manner from the mutational site to the active site residues of DNMT3A. Our computational results provided molecular insights into the pathogenic R882H mutation and advanced the understanding of its mechanism.

Fig. 1. The structure and proposed reaction mechanism of human DNMT3A: (a) the 3D structure constructed via homology modeling; (b) the 2D topological structure with annotated secondary structures; (c) the TOF-determining step in the catalytic cycle; and (d) the TOF-determining transition structure of the methyl transfer step (TDTS).

Fig. 2. The structural variations caused by R882H mutation: (a) the structural deviations between the average structures of WT and R882H-mutated homology holo-models; and (b) a structural representation of the distances between hydroxyl O2′/O3′ atoms and the terminal O (carbonyl oxygen)/OXT (carboxyl oxygen) atoms, and the corresponding statistical t-test results. The unit of distance is the angstrom.

Fig. 3. The hydrogen-bonding network distinctions around SAM upon R882H mutation. The light green dotted lines show hydrogen bonds that are conserved in both WT and R882H-mutated DNMT3A, the red dotted lines represent those that decline in the R882H mutant, and the blue dotted lines represent those that strengthen in the R882H mutant. The line weights accord with the hydrogen-bonding occupancy.

Fig. 4. The ‘protein distortion’ and ‘core interaction’ energies of the MM and QM layers from ONIOM calculations for the methyl transfer step (the energy units are kcal mol; WT DNMT3A is colored blue; R882H-mutated DNMT3A is colored orange).

Fig. 5. The structural representations and hydrogen-bonding patterns in the QM regions of WT-PRS, Mut-PRS, WT-TS, and Mut-TS systems, with WT DNMT3A colored blue and R882H-mutated DNMT3A colored orange; hydrogen bonds are shown as black dashed lines; the hydrogen bonds formed within SAM are highlighted in red circles; SAM is shown as a ball-and-stick model, while other residues are shown as sticks.

Fig. 6. The most probable pathway from site 882 to Arg891 in the apo- and holo-models of the wild type and mutant, from amino acid contact analysis.

Fig. 7. The quasi-“Newton's cradle” mechanism of dynamic information transduction from Arg882/His882 to Arg891 in WT DNMT3A and the R882H mutant. WT DNMT3A is colored blue and the R882H mutant is colored orange.