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，是治疗哮喘和肺纤维化的靶点。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，导致哮喘的特征症状，包括支气管收缩、粘液高分泌和呼吸道水肿。为了了解治疗的特异性以及膜蛋白结构的分子机制和后果，我们建议用电子冷冻显微镜(Cryo-EM)，特别是电子结晶学来研究人类LTC4S。我们的初步研究表明，人类LTC4S可以在一定程度上和数量上过表达和纯化，这使得我们能够重复地诱导二维(2D)结晶。得到的晶体很大，高度有序，投影数据延伸到高于4E的分辨率，可以看到四个跨膜1-螺旋。根据以往的定点突变研究，我们推测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，并为通过生化和结构手段进一步进行功能研究提供基础。在本方案的目标1中，我们将通过低温电子显微镜收集和分析倾斜和未倾斜的2D晶体的数据来确定人类LTC4S的原子结构。该结构将使我们能够得出形成活性部位的位置和残基的结论。在目标2中，我们将结晶人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度特异性。这些信息最终可能被用来探索LTC4S特定的抑制剂。 人类白三烯C4合成酶(LTC4S)的原子模型及其反应机制的深入研究可能有助于更好地理解和治疗一些炎症性疾病，包括哮喘、变应性鼻炎和肺纤维化。特别是，一旦结构可用，就可以探索LTC4S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.