MOLECULAR DYNAMICS SIMULATIONS OF SITE-SPECIFIC PROTEIN-DNA INTERACTIONS: CONFI

位点特异性蛋白质-DNA 相互作用的分子动力学模拟：CONFI

• 批准号: 7601395
• 负责人: LINDA JEN-JACOBSON
• 金额: $ 0.03万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601395
• 关键词: Active Sites; Affect; Area; Attention; Benchmarking; Binding; Biochemical; Catalysis; Characteristics; Charge; Complement Factor B; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Crystallography; DNA; DNA Restriction Enzymes; DNA Sequence; DNA Structure; DNA-Protein Interaction; Data; Deoxyribonuclease EcoRI; Disease; Disruption; Electrostatics; Elements; Fluorescence; Free Energy; Funding; Gene Expression; Genetic; Genetic Recombination; Grant; Heart; Institution; Kinetics; Modeling; Molecular; Molecular Conformation; Motion; NMR Spectroscopy; Pliability; Positioning Attribute; Prevention; Process; Proteins; Range; Regulation; Relaxation; Research; Research Personnel; Resources; Role; Site; Solvents; Source; Specificity; Structure; System; Testing; Thermodynamics; Time; United States National Institutes of Health; Upper arm; Water; cofactor; conformational conversion; improved; insight; interfacial; methylphosphonate; molecular dynamics; mutant; particle; protonation; simulation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein recognition of specific DNA sequences lies at the heart of many normal and disease-related processes, including gene expression and its regulation and genetic recombination. To understand the biophysical principles that govern recognition specificity in protein-DNA interactions, we study the interactions of three restriction endonucleases (EcoRI, BamHI and EcoRV) with their DNA recognition sites, using thermodynamics, kinetics, spectroscopy (NMR, EPR and fluorescence), genetics and computational simulations. Major attention is given to dynamic characteristics of protein-DNA complexes, including binding-dependent conformational transitions in proteins and DNA and conformational-vibrational fluctuations in the complexes. Our enormous accumulation of diverse information on these systems places us in position to pose sharply focused computational questions and to benchmark the computational results against experimental data. This proposal includes the following computational objectives: 1. To use computational simulations to study the interactions of a "promiscuous" mutant EcoRI endonuclease with its DNA recognition site. We will first test if MD simulations (using the AMBER8 suite, with explicit solvent and treatment of long-range interactions by particle-mesh Ewald) can reproduce the relatively subtle differences between mutant and wild-type complexes as determined by x-ray crystallography, to pave the way for simulations of the mutants for which we lack experimental structures. We will then use MD simulations to study the dynamics of the mutant complex, with particular attention to the roles of interfacial water molecules, motions of the protein "arms" that enfold the DNA, and possibly concerted motions in the complex. These studies will include calculation of Debye-Waller B-factors from the MD trajectories, and comparison with the experimental B-factors. Finally, we will do computational time-averaging crystallographic refinement (TACR) using the data on both wild-type and mutant complexes, to study molecular and solvent motions that are physically reasonable and constrained to the envelope defined by the experimental diffraction data. These studies will use the GROMOS package for the MD portion of the TACR. 2. To use MD simulations (with full explicit solvent) to study how conformational dynamics and molecular distortions in the EcoRI-DNA complex are affected by stereospecific methylphosphonate substitutions at two positions of the GAATTC recognition site, for which we have extensive biochemical data. We will use such simulations to study how these derivatives cause (a) disruption of the precise water relay required for catalysis; (b) subtle but crucial alterations in sidechain conformations; (c) prevention of Mg2+ cofactor binding to the active site. These studies should yield new insight into the role of active-site water structure and DNA distortion in the catalytic mechanism. 3. To use MD simulations to study the consequences of electrostatic repulsion (a form of molecular strain) in the active site of the BamHI-DNA complex. Experimental data indicate that the cluster of acidic sidechains in this active site have abnormally high pKa's, that the charge on the E111 sidechain is the critical central controller, and that the consequences of strain include increased molecular motion (broadened dynamic distribution) in the complex. We will first use in silico rebuilding and MD simulation (full explicit solvent) to produce an improved model of the wild-type BamHI-DNA complex, since our biochemical data show the crystal structure lacks essential DNA elements and suffers from distortions due to packing forces. We will then test the effect of protonation of E111 and D94 in the context of wild-type protein and mutant proteins E111A and D94A, examining the following issues: (a) Are the consequences of strain strictly local (e.g., rotamer relaxation) or extended over wider areas of the complex? (b) Are the increased conformational-vibrational motions in strained complexes primarily large motions in a small region or smaller motions over a larger region? (c) Do the protein and DNA undergo concerted motions? We will use a large explicit solvent shell, which allows us to treat protein flexibility and dielectric relaxation explicitly, Finally, we will use molecular dynamics free energy simulations to calculate the pKa shifts of E111 in the context of wild-type and D94A.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 特定 DNA 序列的蛋白质识别是许多正常和疾病相关过程的核心，包括基因表达及其调控和基因重组。为了了解控制蛋白质-DNA 相互作用中识别特异性的生物物理原理，我们利用热力学、动力学、光谱学（NMR、EPR 和荧光）研究了三种限制性内切酶（EcoRI、BamHI 和 EcoRV）与其 DNA 识别位点的相互作用，遗传学和计算模拟。主要关注蛋白质-DNA 复合物的动态特征，包括蛋白质和 DNA 中依赖于结合的构象转变以及复合物中的构象振动波动。我们在这些系统上积累的大量不同信息使我们能够提出尖锐的计算问题，并根据实验数据对计算结果进行基准测试。该提案包括以下计算目标： 1. 使用计算模拟来研究“混杂”突变体 EcoRI 核酸内切酶与其 DNA 识别位点的相互作用。我们将首先测试 MD 模拟（使用 AMBER8 套件，使用显式溶剂并通过粒子网格 Ewald 处理长程相互作用）是否可以重现由 X 射线晶体学确定的突变型和野生型复合物之间相对细微的差异，为我们缺乏实验结构的突变体的模拟铺平道路。然后，我们将使用 MD 模拟来研究突变体复合物的动力学，特别关注界面水分子的作用、包裹 DNA 的蛋白质“臂”的运动，以及复合物中可能的协同运动。这些研究将包括根据 MD 轨迹计算 Debye-Waller B 因子，并与实验 B 因子进行比较。最后，我们将使用野生型和突变型复合物的数据进行计算时间平均晶体学精修（TACR），以研究物理上合理且受限于实验衍射数据定义的包络的分子和溶剂运动。这些研究将使用 GROMOS 软件包来实现 TACR 的 MD 部分。 2. 使用 MD 模拟（使用全显式溶剂）来研究 EcoRI-DNA 复合物中的构象动力学和分子扭曲如何受到 GAATTC 识别位点两个位置上的立体特异性甲基膦酸酯取代的影响，对此我们有大量的生化数据。我们将使用此类模拟来研究这些衍生物如何导致（a）催化所需的精确水传递中断； (b) 侧链构象的微妙但重要的改变； (c) 防止 Mg2+ 辅因子与活性位点结合。这些研究应该会对活性位点水结构和 DNA 扭曲在催化机制中的作用产生新的见解。 3. 使用MD模拟来研究BamHI-DNA复合物活性位点的静电排斥（分子应变的一种形式）的后果。实验数据表明，该活性位点中的酸性侧链簇具有异常高的 pKa，E111 侧链上的电荷是关键的中央控制器，并且应变的后果包括复合物中分子运动的增加（动态分布变宽）。我们将首先使用计算机重建和 MD 模拟（全显式溶剂）来生成野生型 BamHI-DNA 复合物的改进模型，因为我们的生化数据显示晶体结构缺乏必需的 DNA 元素，并且由于包装力而遭受扭曲。然后，我们将在野生型蛋白和突变蛋白 E111A 和 D94A 的背景下测试 E111 和 D94 质子化的影响，检查以下问题： (a) 应变的后果是严格局部的（例如旋转异构体松弛）还是延长的覆盖更广泛的综合体区域？ (b) 应变复合物中构象振动运动的增加主要是小区域内的大运动还是较大区域内的较小运动？ (c) 蛋白质和 DNA 是否进行一致运动？我们将使用大型显式溶剂壳，这使我们能够显式地处理蛋白质柔性和介电弛豫。最后，我们将使用分子动力学自由能模拟来计算 E111 在野生型和 D94A 背景下的 pKa 变化。