Singh Swati, Guruprasad Lalitha
School of Chemistry, University of Hyderabad , Hyderabad 500046, India.
J Phys Chem B. 2017 Jan 19;121(2):365-378. doi: 10.1021/acs.jpcb.6b08433. Epub 2017 Jan 5.
Helicobacter pylori is a primitive Gram-negative bacterium that resides in the acidic environment of the human gastrointestinal tract, and some strains of this bacterium cause gastric ulcers and cancer. DNA methyltransferases (MTases) are promising drug targets for the treatment of cancer and other diseases that are also caused by epigenetic alternations of the genome. The N6-adenine-specific DNA MTase from H. pylori (M. Hpy N6mA) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) to the flipped adenine of the substrate DNA. In this work, we report the sequence analyses, three-dimensional structure modeling, and molecular dynamics simulations of M. Hpy N6mA, when complexed with AdoMet as well as DNA. We analyzed the protein-DNA interactions prominently established by the flipped cytosine and the interactions between protein cofactors in the active site. The comparable orientation of AdoMet in both systems confirms that AdoMet is in a catalytically competent orientation in the bimolecular system that is retained upon DNA binding in the termolecular system of M. Hpy N6mA. In both systems, AdoMet is stabilized in the binding pocket by hydrogen bonding (Thr84, Glu99, Asp122, and Phe123) as well as van der Waals (Ile100, Phe160, Arg104, and Cys76) interactions. We propose that the contacts made by flipped adenine DA6 with Asn138 (N6 and N1 atom of DA6) and Pro139 (N6) and π-stacking interactions with Phe141 and Phe219 play an important role in the methylation mechanism at the N6 position in our N6mA model. Specific recognition of DNA is mediated by residues 143-155, 183-189, 212-220, 280-293, and 308-325. These findings are further supported by alanine scanning mutagenesis studies. The conserved residues in distantly related sequences of the small domain are important in DNA binding. Results reported here elucidate the sequence, structure, and binding features necessary for the recognition between cofactor AdoMet and substrate DNA by the vital epigenetic enzyme, M. Hpy N6mA.
幽门螺杆菌是一种原始的革兰氏阴性细菌，存在于人类胃肠道的酸性环境中，该细菌的一些菌株会导致胃溃疡和癌症。DNA甲基转移酶（MTases）是治疗癌症和其他由基因组表观遗传改变引起的疾病的有前景的药物靶点。来自幽门螺杆菌的N6-腺嘌呤特异性DNA MTase（M. Hpy N6mA）催化辅因子S-腺苷-L-甲硫氨酸（AdoMet）的甲基转移到底物DNA的翻转腺嘌呤上。在这项工作中，我们报告了M. Hpy N6mA与AdoMet以及DNA复合时的序列分析、三维结构建模和分子动力学模拟。我们分析了由翻转的胞嘧啶显著建立的蛋白质-DNA相互作用以及活性位点中蛋白质辅因子之间的相互作用。两个系统中AdoMet的可比取向证实，在双分子系统中AdoMet处于催化活性取向，在M. Hpy N6mA的三分子系统中DNA结合后该取向得以保留。在两个系统中，AdoMet通过氢键（Thr84、Glu99、Asp122和Phe123）以及范德华力（Ile100、Phe160、Arg104和Cys76）相互作用稳定在结合口袋中。我们提出，翻转的腺嘌呤DA6与Asn138（DA6的N6和N1原子）和Pro139（N6）的接触以及与Phe141和Phe219的π-堆积相互作用在我们的N6mA模型中N6位置的甲基化机制中起重要作用。DNA的特异性识别由143-155、183-189、212-220、280-293和308-325位的残基介导。这些发现得到了丙氨酸扫描诱变研究的进一步支持。小结构域远缘相关序列中的保守残基在DNA结合中很重要。此处报道的结果阐明了重要的表观遗传酶M. Hpy N6mA识别辅因子AdoMet和底物DNA所需的序列、结构和结合特征。
