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

项目总结/摘要 脂氧合酶是通过产生脂氧合酶来促进炎症的起始和消退的酶。 特定脂质氧化产物。这些蛋白质停留在胞质溶胶中，并在Ca2+作用下靶向细胞膜 刺激获得底物的地方。本申请的目的是揭示如何构象 状态调节两种人脂氧合酶的膜靶向和底物获取。很长的- 术语的目标是阐明这些人脂氧合酶在构象中的全部分子细节， 脂质底物和小分子抑制剂，以帮助基于结构的药物发现。的理由 拟议的研究是，瞬时结合到膜上的酶应该采用不同的构象， 细胞质和细胞膜两个亚细胞位置之间的分化 疏水残基。本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-脂氧合酶的结构 已确定为"封闭"形式，活性部位无法进入，或不完整的"开放"形式， 关键肽区域，定义了X射线晶体学无法分辨的活性位点。我们的研究方法 结合了战略定点突变体的设计，以解锁和促进一种"开放"构象， 通过动力学和氢氘交换质谱法（HDX-MS）进行验证。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地研究。(2)的作用 在15-脂氧合酶-2的膜底物采集的构象灵活性。脂质类似物 去污剂似乎是15-脂氧合酶-2结晶所必需的。我们会确认脂质结合位点 HDX-MS和X射线晶体学分析了15-LOX-2的基因X射线反射率和掠入射X射线 在朗缪尔槽中与单层结合的15-脂氧合酶-2的衍射将提供最天然的 膜上脂氧合酶的结构数据。这个项目很重要，因为它将描绘 脂质对外周膜结合蛋白构象重塑的作用。我们就会揭开 负责两种人脂氧合酶底物获取的分子决定簇， 设计结合具有改变的动力学常数的底物的人LOX的新变体，（B） 所述酶在脂质类似物去污剂的存在下溶解，和（c）解析结合到所述酶的结构， 脂质类似物去污剂和底物。这项研究是创新的，因为它（a）开发了人类基因的新变体， 5-具有策略突变以促进"开放"形式的脂氧合酶，（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合理反馈，以及（c）将揭示 人脂氧合酶在磷脂膜上相互作用的方向和深度。

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.