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.

项目概要 铁金属蛋白是一大类酶，参与人类必需的许多化学反应 在许多这些蛋白质中，铁硫键在相互作用中发挥着重要的功能作用。 铁活性位点和含硫底物以及作为电子转移中心的[Fe-S]簇。 这些酶参与抗生素的生物合成、硫代谢和细胞呼吸。 因此，了解这些酶在生理相关条件下的催化机制 最近，X 射线自由电子激光器 (XFEL) 已成为回答关键健康相关问题的关键。 是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 不会造成辐射损伤，这使得在条件下实时跟踪催化反应成为可能。 生理条件，提供了对机制的重要见解然而，一个重要的问题是。 XFEL XRD数据分析仍需解决 细化是解决结构的重要一步。 基于从 XRD 获得的电子密度图的蛋白质。 程序使用立体化学约束来帮助结构解决方案并确保最终结构 这些限制对于金属-配体键来说通常不准确，这可能会导致偏差。 细化结果，特别是对于中等分辨率 (~1.6-2.4 Å) 的结构。 立体化学约束不能准确反映金属周围电子密度的电位变化 电荷分布的这种微妙变化可能会导致机械上重要的结构变化。 因此，更准确地包含这些效应的细化至关重要，特别是对于高共价部分 例如 Fe-S 键和团簇的量子力学 (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，从而为标准提供了有吸引力的补充 在本提案中，我将开发和校准一种量子细化方法。 该方法将作为模块集成到晶体学结构分析软件平台 PHENIX 中。 对金属活性位点和紧邻周围的配体使用密度泛函理论 (DFT) 计算 获得晶体学细化过程中使用的梯度，并将以模型铁为基准 然后我将将该方法应用于两种金属蛋白的时间分辨 XFEL XRD 数据， 收集异青霉素 N 合酶 (IPNS) 和耐氧膜结合 [Ni-Fe] 氢化酶 (MBH) 在 O2 反应过程中，这些结构的量子细化将耦合电子的微妙变化。 催化结构变化过程中的结构，给出更准确的结构并阐明机制 IPNS 中异青霉素 N 生物合成的研究和 MBH 中 O2 耐受的机制。 该方法将对未来进一步了解金属酶机制产生重要影响。