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。

基本信息

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
项目类别：
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

项目摘要

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。这些蛋白质存在于细胞质中，并以 Ca2+ 为目标靶向细胞膜。该应用的目的是揭示构象如何发生。状态调节两种人类脂氧合酶的膜靶向和底物获取。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。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 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”形式确定我们的研究方法无法解析定义活性位点的关键肽区域。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。将积极研究有利于“开放”形式的 5-脂氧合酶变体的结构 (2)。 15-脂氧合酶-2 在膜上获取底物时的构象灵活性。去污剂似乎是 15-lipoxygenase-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。 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非常重要，因为它将描绘出膜上脂氧合酶的结构数据。我们将揭示脂质对外周膜结合蛋白构象重塑的作用。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

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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