Structure and function of the membrane protein human leukotriene C4 synthase

描述（申请人提供）：人白三烯C4合酶（LTC 4S）是一种感兴趣的整合膜蛋白，作为治疗支气管哮喘和肺纤维化的靶点。LTC 4S对其底物白三烯（LT）A4具有高度特异性，其与还原型谷胱甘肽结合形成LTC 4，导致哮喘的特征性症状，包括支气管收缩、粘液分泌过多和气道水肿。为了了解分子机制和治疗特异性的后果以及膜蛋白结构，我们建议通过电子冷冻显微镜（cryo-EM），特别是电子晶体学来研究人类LTC 4S。我们的初步研究表明，人LTC 4S可以过表达和纯化到一定程度和数量，这使我们能够可重复地诱导二维（2D）结晶。得到的晶体是大的和高度有序的，和投影数据延伸到优于4 E分辨率可视化四个跨膜1-螺旋。基于先前的定点突变研究，我们假设Arg-51和Tyr-93参与催化，而Ile-27、瓦尔-35、瓦尔-49、Arg-51、Ala-52、Asn-55、Tyr-59、Tyr-93、Tyr-97和Ala- 112形成LTA 4和谷胱甘肽的结合位点。结构数据将提供重要的信息，了解这些残基的安排和作用，并提供了进一步的功能研究的基础，通过生化和结构手段。在本提案的目标1中，我们将通过冷冻EM收集和分析倾斜和未倾斜的2D晶体的数据来确定人类LTC 4S的原子结构。结构将使我们的结论的位置和残基形成的活性位点。在目标2中，我们将结晶人LTC 4S的各种突变体，并进行抑制和激活研究，以了解详细的分子机制和酶的高度特异性。这些信息最终可能用于探索LTC 4S特异性抑制剂。 人类白三烯C4合酶（LTC 4S）的原子模型及其反应机制的深入了解可能会导致更好地理解和治疗一些炎症性疾病，包括哮喘，过敏性鼻炎和肺纤维化。特别是，一旦结构可用，就可以探索LTC 4S特异性抑制剂。

DESCRIPTION (provided by applicant): Human leukotriene C4 synthase (LTC4S) is an integral membrane protein of interest as a target for the treatment of bronchial asthma and pulmonary fibrosis. LTC4S is highly specific for its substrate leukotriene (LT) A4, which it conjugates with reduced glutathione to form LTC4, responsible for the characteristic symptoms of asthma including bronchoconstriction, mucus hypersecretion and edema of the airways. To gain an understanding of the molecular mechanism and the consequences of the specificity for treatment as well as membrane protein structures in general, we propose to study human LTC4S by electron cryo-microscopy (cryo-EM), specifically electron crystallography. Our Preliminary Studies show that human LTC4S can be overexpressed and purified to an extent and in a quantity, which allows us to reproducibly induce two-dimensional (2D) crystallization. The resulting crystals are large and highly ordered, and projection data extending to better than 4E resolution visualizes four transmembrane 1-helices. Based on previous site-directed mutagenesis studies, we hypothesize that Arg-51 and Tyr-93 are involved in catalysis while Ile-27, Val-35, Val-49, Arg-51, Ala-52, Asn-55, Tyr-59, Tyr-93, Tyr-97, and Ala- 112 form the binding site for LTA4 and glutathione. Structural data would provide important information regarding the understanding of the arrangement and role of these residues and provide a basis for further functional studies by both biochemical and structural means. In Aim 1 of this proposal we will determine an atomic structure of human LTC4S by collecting and analyzing data of tilted and untilted 2D crystals by cryo-EM. The structure would allow us conclusions about the location and residues forming the active site. In Aim 2 we will crystallize various mutants of human LTC4S and carry out inhibition and activation studies to understand the detailed molecular mechanism and the high specificity of the enzyme. This information might eventually be used to explore LTC4S specific inhibitors.Project Narrative The atomic model of human leukotriene C4 synthase (LTC4S) and an insight into its reaction mechanism might lead to a better understanding and treatment of a number of inflammatory diseases, including asthma, allergic rhinitis, and pulmonary fibrosis. In particular, LTC4S specific inhibitors could be explored once the structure is available.