Quantum refinement for improvement of metalloenzyme crystal structures

用于改善金属酶晶体结构的量子精炼

• 批准号: 9760561
• 负责人: Kyle D. Sutherlin
• 金额: $ 4.79万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2020-03-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760561
• 关键词: Active Sites; Anabolism; Antibiotics; Atmosphere; Benchmarking; Binding; Catalysis; Cell Respiration; Cell physiology; Cells; Charge; Chemicals; Chemistry; Citric Acid Cycle; Collection; Complex; Computer software; Coupled; Crystallization; Crystallography; Cysteine Metabolism Pathway; Data; Electron Transport; Electrons; Ensure; Enzymes; Evolution; Exposure to; Future; Generations; Goals; Health; Human; Hydrogenase; Iron; Iron-Sulfur Proteins; Lead; Ligands; Maps; Mechanics; Membrane; Metalloproteins; Metals; Methodology; Methods; Modeling; Nature; Penicillins; Physiological; Play; Procedures; Proteins; Radiation induced damage; Ralstonia eutropha; Reaction; Resolution; Roentgen Rays; Role; Rubredoxins; Sampling; Site; Source; Structure; Sulfur; Sulfur Metabolism Pathway; Superoxides; System; Temperature; Theoretical Studies; Time; Valine; Work; X ray diffraction analysis; Xray Emission Spectroscopy; base; cofactor; density; design; electron density; electronic structure; enzyme mechanism; experimental study; improved; insight; iron hydrogenase; isopenicillin N; metalloenzyme; method development; molecular mechanics; novel; oxidation; quantum; restraint; spectroscopic data; theories; x-ray free-electron laser

Project Summary Iron metalloproteins are a large class of enzymes that are involved in many chemistries essential to human health. In many of these proteins, iron sulfur bonds play an important functional role, both in interactions between the iron active site and a sulfur-containing substrate and in [Fe-S] clusters that serve as electron transfer centers. Such enzymes are involved in the biosynthesis of antibiotics, sulfur metabolism, and cellular respiration. Understanding these enzymes’ mechanisms of catalysis under physiologically relevant conditions is thus essential for answering critical health-related questions. Recently, X-ray free electron lasers (XFELs) have emerged as a way to collect time-resolved X-ray diffraction (XRD) data on enzyme crystals at room temperature without causing radiation damage. This has made it possible to follow catalytic reactions in real time under physiological conditions, providing significant insight into mechanism. However, an important issue with the analysis of XFEL XRD data still needs to be resolved. Refinement is an important step in solving the structure of a protein based on the electron density map obtained from XRD. Standard crystallographic refinement procedures use stereochemical restraints to aid in the structure solution and ensure that the final structure is chemically reasonable. These restraints are often not accurate for metal-ligand bonds, which can bias the refinement results, especially for structures solved to medium resolution (~1.6-2.4 Å). Further, these stereochemical restraints do not accurately reflect potential changes in the electron density around the metal site. Such subtle changes in charge distribution can lead to structural changes that are mechanistically important; thus, refinement that more accurately includes these effects is essential, especially for highly covalent moieties such as Fe-S bonds and clusters. Quantum mechanical (QM) calculations can more accurately describe the electron density of covalent metal-ligand bonds and clusters, thus providing an attractive supplement to standard refinement procedures. In this proposal, I will be developing and calibrating a quantum refinement method for integration as a module into the crystallography structure analysis software platform PHENIX. The method will use density functional theory (DFT) calculations on the metal active site and immediately surrounding ligands to obtain the gradient used in the crystallographic refinement procedure, and will be benchmarked on model iron- sulfur proteins. I will then apply the method to time-resolved XFEL XRD data on two metalloproteins, isopeninicillin N synthase (IPNS) and the O2-tolerant membrane-bound [Ni-Fe] hydrogenase (MBH) collected during their O2 reactions. Quantum refinement of these structures will couple the subtle changes in electronic structure during catalysis to changes in structure, giving more accurate structures and elucidating the mechanism of isopenicillin N biosynthesis in IPNS and the mechanism of O2-tolerance in MBH. The development of this method will have important implications for understanding further metalloenzyme mechanisms in the future.

项目摘要 铁金属蛋白是一大类酶，参与人体必需的许多化学反应， 健康在许多这些蛋白质中，铁硫键起着重要的功能作用，无论是在相互作用之间 铁活性中心和含硫底物以及作为电子转移中心的[Fe-S]簇中。 这些酶参与抗生素的生物合成、硫代谢和细胞呼吸。 因此，了解这些酶在生理学相关条件下的催化机制， 对于回答关键的健康相关问题至关重要。最近，X射线自由电子激光器（XFEL）已经 作为一种在室温下收集酶晶体时间分辨X射线衍射（XRD）数据的方法出现 而不会造成辐射损伤。这使得有可能在真实的时间内跟踪催化反应， 生理条件下，提供重要的洞察机制。然而，一个重要的问题是， XFEL XRD数据的分析仍然需要解决。精化是求解结构的重要步骤， 基于从XRD获得的电子密度图的蛋白质。标准晶体学精修 程序使用立体化学限制来帮助结构解决方案，并确保最终的结构是 化学上合理的。这些限制对于金属-配体键通常是不准确的，这会使金属-配体键的结合偏向于金属-配体键。 精细化结果，特别是对于中等分辨率（~1.6-2.4 μ m）的结构。此外，这些 立体化学约束不能准确地反映金属周围电子密度的潜在变化 绝佳的价钱电荷分布的这种微妙变化可以导致在机械上重要的结构变化; 因此，更精确地包括这些效应的改进是必要的，特别是对于高度共价的部分 如Fe-S键和团簇。量子力学（QM）计算可以更准确地描述 共价金属配体键和簇的电子密度，从而提供了一个有吸引力的补充标准 细化程序。在这个提议中，我将开发和校准一种量子细化方法， 作为一个模块集成到晶体学结构分析软件平台PHENIX中。该方法将 使用密度泛函理论（DFT）计算金属活性位点和直接围绕的配体， 获得晶体学细化程序中使用的梯度，并将以模型铁为基准- 硫蛋白然后，我将该方法应用于两种金属蛋白的时间分辨XFEL XRD数据， 异青霉素N合酶（IPNS）和O2耐受性膜结合[Ni-Fe]氢化酶（MBH）收集 在O2反应中。这些结构的量子细化将耦合电子中的微妙变化， 在催化过程中结构的变化，给出了更准确的结构，并阐明了机理 IPNS中异青霉素N生物合成的影响以及MBH中耐氧机制的研究。发展这个 方法将有重要的意义，了解进一步的金属酶的机制在未来。