Molecular dynamics studies of DNA repair and cell signaling enzymes
The focus of this proposal is to increase our understanding of the working mechanisms of the base-excision DNA repair (BER) and in cell nutrient sensing enzymes, by studying human 8-oxoguanine DNA glycosylase and sestrin 2. 1. Human 8-oxoguanine DNA glycosylase (hOGG1) is a key BER enzyme searching and removing DNA lesions caused by oxidation damage to guanine, known as 8-oxoguanine (oxoG). In this project, we aim to elucidate detailed mechanisms of (1) how the enzyme locates lesions in vast excess of normal DNA and (2) multistep reaction pathways leading to the catalytic removal of oxoG. In collaboration with Prof. Gregory Verdine (Harvard University, USA), my lab has been performing computer simulation studies based on the high-resolution structures representing hOOG1 in action determined by the Verdine lab. During the previous allocation periods, we have performed μs-long MD simulations and characterized the dynamics of the enzyme in complex with normal and lesion bases. In the coming allocation period, we will continue the study (1) to determine the complete free energy landscape of the conformational change pathways of base extrusion out of DNA helix and (2) characterize the effects of DNA damage on the determined free energy landscape, by applying the string free energy simulation methods developed recently by my lab. The proposed study will yield new insight into the lesion recognition mechanism of this important enzyme. This part of the proposal replaces our existing allocation at NSC as the support from NSC is disconnected in near future. 2. Sestrins are proteins that attenuate aging by reducing reactive oxygen species inhibiting mTOR complex 1 (mTORC1). Recently, Sestrin became implicated in nutrient sensing and signaling. In collaboration with Profs. Uhn-Soo Cho and Jun Hee Lee (University of Michigan, Ann Arbor, USA), we aim to understand the molecular basis of how Sestrin function is modulated by amino acids and post-translational modification, e.g. phosphorylation. Former cell biology and X-ray crystallography studies unveiled the mechanism of how Sestrin2 suppresses mTORC1, but could not address how small molecules like leucine and protein phosphorylation affect its activity. In contrast to the recent report speculating that Sestrin undergoes drastic conformational changes upon leucine binding, Cho lab’s recent structure of Sestrin2 disproves this model. In this proposal, we will carry out molecular dynamics simulations to gain insights into how nutrient ligands (e.g. leucine) and nutrient-induced protein phosphorylation affect Sestrin2 structure and dynamics. In addition, we will study the catalytic mechanism of the enzyme (i.e., its oxidoreductase activity) by the quantum mechanical and molecular mechanical (QM/MM) simulations. This integrated approach will reveal novel regulatory mechanisms on how diverse nutrient and stress signals are transmitted through Sestrin2. The proposed simulations are to be performed in explicit water and require continued support from SNIC. In particular, the proposed string method and QM/MM simulations are demanding because they require an ensemble of MD simulations running concurrently, and because of the long running time needed to obtain converged thermodynamic averages.