Molecular dynamics simulations of the substrate recognition and specificity of protein and DNA methyltransferases

In the last grant period, we successfully established different molecular dynamic simulation (MD) approaches to investigate the mechanism of protein and DNA methyltransferases and showed that the approach provides highly relevant data helping to understand and rationalize biochemical processes. Based on this, we continue our work in four work packages 1) We plan to validate MD finding of the last grant period to improve steered MD (sMD) simulations of peptide association processes using stopped-flow kinetic experiments. Moreover, we plan to investigate if hairpin peptides can be used as PKMT inhibitors. 2) We plan to apply our MD technology on the NSD2 enzyme, which plays important roles in controlling development and cancer. Based on specificity data, we plan to improve the MD based specificity prediction and at the same time understand the molecular mechanisms underlying a 100-fold increased methylation rate of an NSD2 super-substrate that has already been identified. 3) We will investigate the mechanistic basis of the dual specificity of the PRDM9 enzyme by biochemical methods and MD simulations. This includes the investigation of the effects of cancer mutations of PRDM9 and study the mechanism allowing the enzyme to approach two distinct target sites on the H3-tail. 4) We plan to develop novel MD and sMD based methods to investigate the sequence specific interaction of DNMTs with DNA. They will be applied to identify the molecular mechanisms of the distinct effects of flanking sequences on the activities of the DNMT3A and DNMT3B enzymes, as well as on the medically highly relevant DNMT3A R882H mutant. Our project addresses a fundamental question relevant for the entire group of enzymes introducing nucleic acid and protein modifications by addressing the principles of substrate selection and control of enzyme activity.