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.

项目概要/摘要 脂氧合酶是通过产生脂氧合酶来促进炎症的引发和消退的酶。 特定的脂质氧化产品。这些蛋白质位于细胞质中，并在 Ca2+ 作用下靶向细胞膜 获得底物的刺激。该应用程序的目的是揭示构象如何 状态调节两种人类脂氧合酶的膜靶向和底物获取。长- 术语目标是阐明这些人类脂氧合酶在构象中的完整分子细节 脂质底物和小分子抑制剂有助于基于结构的药物发现。理由如下： 拟议的研究是瞬时与膜结合的酶应该采用不同的构象 通过掩埋或暴露区分细胞质和细胞膜的两个亚细胞位置 疏水性残基。该项目的目标将通过两个具体目标来实现：（1）阐明 人 5-脂氧合酶的催化活性和开放形式。人类 5-脂氧合酶的先前结构 已确定为“封闭”形式，活动站点不可访问，或以不完整的“开放”形式确定 定义 X 射线晶体学无法解析的活性位点的关键肽区域。我们的研究方法 结合了战略定点突变体的设计来解锁和促进“开放”构象，这将 通过动力学和氢化氘交换质谱 (HDX-MS) 进行验证。高分辨率 将积极研究有利于“开放”形式的 5-脂氧合酶变体的结构。 （二）作用 15-脂氧合酶-2 在膜上获取底物时的构象灵活性。脂质类似物 去垢剂似乎是 15-脂氧合酶-2 结晶所必需的。我们将确认脂质结合位点 通过 HDX-MS 和 X 射线晶体学分析 15-LOX-2。 X 射线反射率和掠入射 X 射线 15-lipoxygenase-2 与 Langmuir 槽中的单层结合的衍射将提供最天然的 膜上脂氧合酶的结构数据。拟议的项目意义重大，因为它将描绘 脂质对外周膜结合蛋白构象重塑的作用。我们将揭晓 负责两种人脂氧合酶底物获取的分子决定因素：(a) 人类 LOX 的新变体的设计，其结合具有改变的动力学常数的底物，(b) HDX-MS 在脂质类似物去垢剂存在下的酶，以及（c）解析结合到的酶的结构 脂质类似物去垢剂和底物。这项研究具有创新性，因为它 (a) 开发了人类的新变体 具有策略突变以促进“开放”形式的 5-脂氧合酶，(b) 结合 HDX-MS 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 人脂氧合酶在磷脂膜上相互作用的方向和深度。

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