Dissociation pathways of the p53 DNA binding domain from DNA and critical roles of key residues elucidated by dPaCS-MD/MSM

The dissociation process of the DNA binding domain of p53 (p53-DBD) from a DNA duplex that contains the consensus sequence, which is the specific target of p53-DBD, was investigated by a combination of dissociation parallel cascade selection molecular dynamics (dPaCS-MD) and the Markov state model (MSM). Based on an all-atom model including explicit solvent, we first simulated the p53-DBD dissociation processes by 75 trials of dPaCS-MD, which required an average simulation time of 11.2 ± 2.2 ns per trial. By setting the axis of the DNA duplex as the Z-axis and the binding side of p53-DBD on DNA as the + side of the X-axis, we found that dissociations took place along the +X and −Y directions (−Y directions) in 93% of the cases, while 7% of the cases moved along +X and +Y directions (+Y directions). Toward the −Y directions, p53-DBD dissociated first from the major groove and then detached from the minor groove, while unbinding from the minor groove occurred first in dissociations along the +Y directions. Analysis of the free energy landscape by MSM showed that loss of the minor groove interaction with p53-DBD toward the +Y directions incurred a relatively high energy cost (1.1 kcal/mol) upon a critical transition, whereas major groove detachment more frequently occurred with lower free energy costs. The standard binding free energy calculated from the free energy landscape was −10.9 ± 0.4 kcal/mol, which agrees with an experimental value of –11.1 kcal/mol. These results indicate that the dPaCS-MD/MSM combination can be a powerful tool to investigate dissociation mechanisms of two large molecules. Minor groove binding is mainly stabilized by R248, identified as the most important residue that tightly binds deep inside the minor groove. Analysis of the p53 key residues for DNA binding indicates high correlations with cancer-related mutations, confirming that impairment of the interactions between p53-DBD and DNA can be frequently related to cancer.