Deciphering the Molecular Mechanistic of the H3K27M Mutation and PRC2 complex binding in Diffuse Intrinsic Pontine Glioma (DIPG)

Abstract

DIPG, a fatal pediatric brainstem tumor with a 0% survival rate, is hindered by its inoperable location, limiting research. It is driven by the H3K27M mutation in histone 3, causing structural changes in PRC2 and aberrant gene activation that leads to the childhood brainstem tumors. This study explores the H3K27M mutation's impact on PRC2 and gene regulation. The methodology employed includes extensive molecular dynamics (MD) simulations of the Mutant peptide (H3K27M) – PRC2 complex and Wild Type peptide (H3K27me3) – PRC2 complex. Subsequent to MD simulations, various analyses including RMSD and RMSF plots, counts and stability of Peptide-Protein interactions, and binding free energy analysis were conducted. Results unveiled that, in the absence of EED and SUZ12, the protein displayed heightened RMSD values spanning from 0-20 Å, indicative of instability. However, upon binding with the peptides (mutant or wild-type) and EED, SUZ12, the RMSD plot displayed stability in the PRC2 complex. Notably, the H3K27M-PRC2 complex exhibited higher RMSD values ranging from 1.95-5 Å compared to the H3K27me3-PRC2 complex, which maintained a range of 0.9 – 1.8 Å. Furthermore, the simulation revealed the straightening of the SBD subdomain of PRC2 in complex with the mutant peptide, a typically bent configuration in the active state of PRC2. This alteration may have contributed to PRC2 inactivity. Importantly, this analysis highlighted the pivotal role of ASN-668 in EZH2 for robust binding of H3K27M to PRC2, resulting in stable bonding and PRC2 inactivation. Moreover, interactions between SRM and SET-I regions of EZH2, involving Lys_660 and Valine_657 of SET-I and Phe_145, ASN_142, and Leu_149 of SRM, were thought to activate the lysine-binding substrate. However, the mutant peptide disrupted these interactions, particularly by interacting with Lys_660 of Set-I, pulling K-660 away from SRM’s residue and preventing SET-I from becoming active which consequently inhibit lysine binding. This is potentially crucial in deactivating the PRC2 complex. The presented binding free energy calculations validated the hypothesis present in the literature that the H3K27M peptide exhibits stronger binding affinity to the PRC2 complex, as reflected by a more negative ΔG_bind value i.e. -126.3079 kcal/mol as compared to the wild-type H3K27me3 i.e. -42.3238 kcal/mol. This shift in binding energy underscores the mutation's disruptive effect on the normal functioning of the PRC2 complex, implying a potential role in aberrant gene regulation. In summary, the presented computational exploration yields valuable insights into the structural and functional consequences of the H3K27M mutation within the PRC2 complex. These findings advance our understanding of epigenetic deregulation in cancer and may offer promising avenues for future therapeutic interventions.