MOLECULAR DYNAMICS SIMULATIONS TO UNDERSTAND THE EFFECTS OF ACTIVE-SITE MUTATIO

通过分子动力学模拟了解活性位点突变的影响

• 批准号: 8364310
• 负责人: LINDA JEN-JACOBSON
• 金额: $ 0.11万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364310
• 关键词: Active Sites; Address; Affect; Affinity; Alanine; Amber; Binding; Biomedical Research; Catalysis; Charge; Complex; Coupling; DNA; Data; Electrostatics; Enzymes; Excision; Funding; Future; Grant; High Performance Computing; Ions; LGLA; Laboratories; Metal Ion Binding; Molecular Conformation; Mutation; National Center for Research Resources; Oxygen; Play; Positioning Attribute; Principal Investigator; Proteins; Research; Research Infrastructure; Resources; Role; Running; Side; Site; Source; Specificity; Structure; System; Thermodynamics; Titrations; Type II site-specific deoxyribonuclease; United States National Institutes of Health; Water; abstracting; cofactor; cost; divalent metal; inorganic phosphate; interest; molecular dynamics; mutant; programs; protonation; simulation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract: All type II restriction endonucleases contain a cluster of active-site Glu and Asp residues, which are responsible for coordination of the catalytic cofactor Mg2+. The site for metal ion binding is not assembled completely and precisely until the enzyme binds to its specific DNA recognition sequence. This provides a mechanism for enhancing specificity by coupling recognition to catalysis. At the specific EcoRI-DNA interface, this active-site cluster includes D91, E111, and the scissile phosphate oxygen, located at the GPAATTC position, within the EcoRI specific recognition sequence. D59 and E144 are also located in the vicinity of the acidic active-site cluster but do not play a role in coordinating Mg2+. In addition, two basic side-chain residues are located near this acidic cluster namely, R145 and K113. Apposition of the negative charges in the active-site cluster creates strong electrostatic repulsion, which can be relieved by the addition of divalent metal, or by protonation of active-site residues. Thus, large enhancements in binding affinity (~500-fold in KA) are observed upon removal of negative charge from the protein (by mutation of active-site acidic residues to alanine), from the DNA (by removal of the scissile phosphate), by addition of the divalent metal ion Ca2+, which acts as a non-catalytic mimic of Mg2+, or by titration to pH below 6. Current Interests: We want to extend our understanding of the role of active-site repulsion in the structural, dynamic, and energetic aspects of specific EcoRI-DNA complex formation. Specifically, we ask 1) what are the rotameric positions and the degree of atomic fluctuation of the charged active-site residues in the free enzyme, and in the enzyme-DNA complex in the presence and absence of divalent metal, 2) what are the electrostatic potentials at the active-site in all three of these structures, 3) how does mutation of charged residues to a neutral residue, or to a residue of the opposite charge, affect the rotameric conformation, atomic fluctuation, and electrostatic potentials of active-site residues in all three structures, and 4) how is the water structure at or near the active-site affected by electrostatic perturbation in all three structures? Justification for Allocation: In order to address the questions above, we will use the Amber suite of programs to perform MD simulations and electrostatic potential calculations on wild-type and mutant EcoRI complexes, in the presence and absence of DNA and divalent metal. Due to the size of these systems and the need to run long simulations in order to see possible conformational fluctuations, we believe the XT3 platform will suit our needs. We recognize that the XT3 Cray system will be decommissioned on March 31st, 2010, so we include in our request an allocation on the People SGI Altix 7400 system in order to transition to this platform in the future. Data obtained from these studies, along with the results of rigorous thermodynamic analyses performed in our laboratory, will allow us to better understand the structural, energetic, and dynamic role of the acidic cluster of residues at the EcoRI-DNA active-site, and allow us to compare this system with the active-site of other type II restriction endonucleases having similar but slightly different, active-site geometries.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 摘要：所有 II 型限制性内切酶均含有活性位点 Glu 和 Asp 残基簇，它们负责催化辅因子 Mg2+ 的协调。在酶与其特定的 DNA 识别序列结合之前，金属离子结合位点并未完全且精确地组装。这提供了一种通过将识别与催化耦合来增强特异性的机制。在特定的 EcoRI-DNA 界面上，该活性位点簇包括 D91、E111 和可裂解的磷酸氧，位于 EcoRI 特异性识别序列内的 GPAATTC 位置。 D59 和 E144 也位于酸性活性位点簇附近，但在 Mg2+ 配位中不发挥作用。此外，两个碱性侧链残基位于该酸性簇附近，即 R145 和 K113。活性位点簇中负电荷的并置会产生强烈的静电排斥，可以通过添加二价金属或通过活性位点残基的质子化来缓解。因此，在从蛋白质（通过将活性位点酸性残基突变为丙氨酸）、从 DNA（通过去除可裂解磷酸盐）中去除负电荷后，观察到结合亲和力的大幅增强（KA 中约 500 倍），通过添加二价金属离子 Ca2+（它充当 Mg2+ 的非催化模拟物），或通过滴定至 pH 值低于 6。当前兴趣：我们希望扩展我们对活性位点作用的理解特定 EcoRI-DNA 复合物形成的结构、动态和能量方面的排斥。具体来说，我们询问 1) 游离酶中以及存在和不存在二价金属的酶-DNA 复合物中带电活性位点残基的旋转异构位置和原子波动程度是多少，2) 什么是带电活性位点残基？所有这三种结构中活性位点的静电势，3）带电残基突变为中性残基或相反电荷的残基，如何影响活性的旋转异构体构象、原子涨落和静电势地点所有三个结构中的残留物，以及4）所有三个结构中活性位点处或附近的水结构如何受到静电扰动的影响？分配理由：为了解决上述问题，我们将使用 Amber 程序套件在存在和不存在 DNA 和二价金属的情况下对野生型和突变型 EcoRI 复合物进行 MD 模拟和静电势计算。由于这些系统的规模以及需要运行长时间模拟才能看到可能的构象波动，我们相信 XT3 平台将满足我们的需求。我们认识到 XT3 Cray 系统将于 2010 年 3 月 31 日退役，因此我们在请求中纳入了 People SGI Altix 7400 系统的分配，以便将来过渡到该平台。从这些研究中获得的数据，以及我们实验室进行的严格热力学分析的结果，将使我们能够更好地了解 EcoRI-DNA 活性位点酸性残基簇的结构、能量和动态作用，并允许我们将该系统与其他具有相似但略有不同的活性位点几何形状的 II 型限制性内切核酸酶的活性位点进行比较。