Structure and function of the membrane protein human leukotriene C4 synthase

描述（由申请人提供）：人白三烯C4合成酶（LTC4S）是一种完整的膜蛋白，可作为支气管哮喘和肺纤维化治疗的靶点。LTC4S对其底物白三烯（LT） A4具有高度特异性，它与还原型谷胱甘肽结合形成LTC4，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，我们建议使用电子冷冻显微镜（cryo-EM），特别是电子晶体学来研究人类LTC4S。我们的初步研究表明，人类LTC4S可以在一定程度和数量上过表达和纯化，这使我们能够重复地诱导二维（2D）结晶。所得晶体大且高度有序，投影数据扩展到优于4E分辨率，可以看到四个跨膜1螺旋。基于之前的定点诱变研究，我们假设Arg-51和Tyr-93参与了催化作用，而Ile-27、Val-35、Val-49、Arg-51、Ala-52、Asn-55、Tyr-59、Tyr-93、Tyr-97和Ala- 112形成了LTA4和谷胱甘肽的结合位点。结构数据将为了解这些残基的排列和作用提供重要信息，并为进一步通过生化和结构手段进行功能研究提供基础。在本提案的目标1中，我们将通过低温电镜收集和分析倾斜和未倾斜二维晶体的数据来确定人类LTC4S的原子结构。这种结构可以让我们得出活性位点的位置和残基形成的结论。在Aim 2中，我们将结晶人类LTC4S的各种突变体，并进行抑制和激活研究，以了解该酶的详细分子机制和高特异性。这些信息可能最终用于探索LTC4S特异性抑制剂。项目的叙述

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.