Conformational Flexibility of Lipoxygenases and its Role in Regulation and Substrate Acquisition.

脂氧合酶的构象灵活性及其在调节和底物获取中的作用。

• 批准号: 10292333
• 负责人: Nathaniel Gilbert
• 金额: $ 44.4万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292333
• 关键词: Active Sites; Adopted; Allergic rhinitis; Amino Acids; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Asthma; Binding; Binding Proteins; Binding Sites; Biological; Biophysics; Burial; Cause of Death; Choline; Chronic; Collaborations; Colorado; Complex; Crystallization; Cytoplasm; Cytosol; Data; Detergents; Deuterium; Development; Enzymes; Feedback; Funding; Generations; Goals; Health; Human; Hydrogen; Hydrophobic Interactions; Hydrophobicity; Incidence; Infection; Inflammation; Inflammatory; Institution; Iron; Kinetics; Leukotrienes; Ligands; Link; Lipid Bilayers; Lipid Binding; Lipids; Lipoxygenase; Lipoxygenase 2; Location; Louisiana; Mass Spectrum Analysis; Membrane; Methods; Molecular; Molecular Conformation; Molecular Machines; Mutation; Names; Oxygen; Partner in relationship; Pathway interactions; Peptides; Peripheral; Phase; Phospholipids; Play; Polyunsaturated Fatty Acids; Positioning Attribute; Protein Isoforms; Proteins; Regulation; Research; Resolution; Rest; Roentgen Rays; Role; Sampling; Series; Site; Structure; Students; Techniques; Temperature; Testing; Tissues; Training; Universities; Variant; X ray diffraction analysis; X-Ray Crystallography; analog; base; design; drug discovery; enzyme structure; flexibility; inhibitor/antagonist; innovation; mimetics; monolayer; mutant; oxidation; oxidative damage; response; small molecule inhibitor; undergraduate student

Project Summary/Abstract Lipoxygenases are enzymes that contribute to the initiation and resolution of inflammation by generating specific lipid oxygenation products. These proteins rest in the cytosol and target to the membrane upon Ca2+ stimulation where substrate is acquired. The objective of this application is to revel how the conformational state regulates membrane targeting and substrate acquisition for two human lipoxygenase enzymes. The long- term goal is to elucidate the full molecular details of these human lipoxygenases in conformations bound to lipid substrate and small molecule inhibitors to aid in structure-based drug discovery. The rationale for the proposed research is enzymes that bind to the membrane transiently should adopt distinct conformations for differentiation between the two subcellular locations of the cytoplasm and the membrane by burial or exposure of hydrophobic residues. The objective of this project will be accomplished by two specific aims: (1) Elucidating the catalytically active and open form of human 5-lipoxygenase. Previous structures of human 5-lipoxygenase have been determined in a “closed” form with the active site inaccessible or in an incomplete “open” form with key peptide regions that define the active site unresolved by X-ray crystallography. Our research approach combines the design of strategic site-directed mutants to unlock and promote an “open” conformation that will be validated by kinetics and Hydroden Deuterium eXchange by Mass Spectrometry (HDX-MS). High-resolution structures of variants of 5-lipoxygenase that favor an “opened” form will be aggressively pursued. (2) The role of conformational flexibility in substrate acquisition at the membrane by 15-lipoxygenase-2. Lipid-analog detergents appear to be necessary for crystallization of 15-lipoxygenase-2. We will confirm lipid-binding sites on 15-LOX-2 through HDX-MS and X-ray crystallography. X-ray reflectivity and grazing incidence X-ray diffraction of 15-lipoxygenase-2 bound to a monolayer in a Langmuir trough will provide the most native structural data of a lipoxygenase at the membrane. The proposed project is significant because it will delineate the role lipids have on the conformational remodeling of a peripheral membrane-binding protein. We will reveal the molecular determinants that are responsible for substrate acquisition of two human lipoxygenases by (a) the design of new variants of human LOXs that bind substrate with altered kinetic constants, (b) HDX-MS of the enzymes in the presence of lipid-analog detergents, and (c) solve the structure of the enzymes bound to lipid-analog detergents and substrate. This study is innovative because it (a) develops new variants of human 5-lipoxygenase with strategic mutations to promote an “opened” form, (b) combines the methods of HDX-MS to provide rationale feedback of conformational flexibility to our X-ray crystallography efforts, and (c) will reveal the orientation and the depth of interactions of a human lipoxygenase at the phospholipid membrane.

项目摘要/摘要 脂氧酶是通过产生主动性和分辨率的酶 特定的脂质氧合产物。这些蛋白质在Ca2+上均位于细胞质中并靶向膜 获取底物的刺激。该应用的目的是陶醉于构象如何 国家调节两种人脂氧酶酶的膜靶向和底物采集。长期 术语目标是以与 脂质底物和小分子抑制剂，以帮助基于结构的药物发现。理由 拟议的研究是瞬时与膜结合的酶，应采用不同的构象 通过埋葬或暴露，细胞质的两个亚细胞位置与膜之间的区分 疏水残差。该项目的目的将通过两个具体目标来完成：（1）阐明 人类5-脂氧酶的催化活性和开放形式。人类5-脂氧酶的先前结构 已经以“封闭”形式确定活跃位点无法访问或以不完整的“打开”形式确定 X射线晶体学无法分辨的活性位点的关键肽区域。我们的研究方法 结合战略性地点定向突变体的设计，以解锁和促进“开放”构象 可以通过质谱法（HDX-MS）的动力学和Hydroden氘交换来验证。高分辨率 有利于“开放”形式的5-脂氧合酶变体的结构将被积极追求。 （2）角色 15-脂氧合酶-2在膜上采集底物的构象柔韧性。脂质分析 洗涤剂似乎对于15-脂氧酶-2的结晶是必需的。我们将确认脂质结合位点 在15-LOX-2上通过HDX-MS和X射线晶体学。 X射线反射率和放牧事件X射线 在Langmuir麻烦中与单层结合的15-脂氧合酶-2的衍射将提供最天然的 Lipoxygogasy酶在膜上的结构数据。拟议的项目很重要，因为它将描绘 脂质在周围膜结合蛋白的构象重塑中具有作用。我们将透露 负责通过（a）对两种人Lipoxygoxygasy酶进行底物获取的分子决定剂 与动力学常数改变底物的人Lox的新变体的设计，（b）HDX-MS的HDX-MS 酶在脂质 - 肛门dexts存在下，（c）求解与 脂质 - 分析dexts和底物。这项研究具有创新性，因为它（a）开发了人类的新变体 具有战略突变以促进“打开”形式的5-脂氧合酶，（b）结合了HDX-MS的方法 为我们的X射线晶体学工作提供构象灵活性的基本原理反馈，并且（c）将揭示 人脂氧酶在磷脂膜上的方向和相互作用的深度。

项目成果

Untangling the web of 5-lipoxygenase-derived products from a molecular and structural perspective: The battle between pro- and anti-inflammatory lipid mediators.

• DOI: 10.1016/j.bcp.2021.114759
• 发表时间: 2021-11
• 期刊: Biochemical pharmacology
• 影响因子: 5.8
• 作者: Gilbert NC;Newcomer ME;Werz O
• 通讯作者: Werz O

Investigating membrane-binding properties of lipoxygenases using surface plasmon resonance.

使用表面等离子体共振研究脂氧合酶的膜结合特性。

• DOI: 10.1016/j.bbrc.2023.05.066
• 发表时间: 2023
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Rohlik,DeniseL;Patel,Ethan;Gilbert,NathanielC;Offenbacher,AdamR;Garcia,BrandonL
• 通讯作者: Garcia,BrandonL

Allosteric Activation of 15-Lipoxygenase-1 by Boswellic Acid Induces the Lipid Mediator Class Switch to Promote Resolution of Inflammation.

• DOI: 10.1002/advs.202205604
• 发表时间: 2023-02
• 期刊: ADVANCED SCIENCE
• 影响因子: 15.1
• 作者: Boerner, Friedemann;Pace, Simona;Jordan, Paul M.;Gerstmeier, Jana;Gomez, Mario;Rossi, Antonietta;Gilbert, Nathaniel C.;Newcomer, Marcia E.;Werz, Oliver
• 通讯作者: Werz, Oliver