Molecular Mechanism of the Parkinson's Disease-associated protein LRRK2

项目摘要： LRRK 2是一种功能未知的双酶多结构域蛋白。LRRK 2的突变与 几种不同的疾病，包括帕金森病，炎症性肠病，结核病麻风病， 和老年痴呆症 本研究计划的目的是通过使用LRRK 2来确定LRRK 2作用的分子机制。 生物化学、生物物理学和化学生物学方法。我们已经证明，LRRK 2的ROC是一个真正的 PD相关突变通过损害GT3活性使其处于持续激活状态， 经历由疾病相关突变损害的核苷酸依赖性构象变化。 拟议的研究试图了解ROC结构域中的这些构象变化如何调节蛋白质的表达。 LRRK 2的整体结构和活性。这三个目标是： 目的1：确定LRRK 2的结构及其构象动力学。 a）确定在不同核苷酸结合状态下由ROC引起的LRRK 2中的结构变化，以及那些 携带PD相关突变的人我们初步的单粒子数据揭示了2种构象 其可以表示“开”和“关”状态。我们将在拟议的研究中定义这些结构。B）定义 LRRK 2的原子结构和相互作用通过使用X射线晶体学，这是定义原子的理想方法 和债券已经确定了产生全长LRRK 2衍射晶体的条件。（三）定义 使用NMR分析ROC中残基R1398与GTP的γ-磷酸的动态相互作用，这是调节 LRRK 2活性机制。 目标2：确定LRRK 2的活动。 a）使用测量携带PD-1的构建体的GT3活性来定义GT3活性的调节。 突变、磷酸化模因和工程化构象约束二硫键。B）确定 通过测量构象稳定的LRRK 2的激酶活性来确定LRRK 2的激酶活性和非活性构象 结构。c）表征细胞模型中LRRK 2的不同构象的活性和定位。 目的3：确定LRRK 2的构象-活性关系。 a）通过采用高通量化学筛选，使用化学手段调节LRRK 2构象 和化学生物学方法。B）工程化生物制剂和潜在的治疗性抗体，用于捕获 LRRK 2的构象通过使用噬菌体展示筛选方法。

Project Summary: LRRK2 is a dual enzyme multidomain protein of unknown function. Mutations in LRRK2 has been associated several different diseases including Parkinson’s disease, inflammatory bowel disease, tuberculosis leprosy, and Alzheimer’s disease. The objective of this research program is to determine the molecular mechanisms of action of LRRK2 by using biochemical, biophysical, and chemical biology methods. We have shown that ROC of LRRK2 is a bona fide GTPase, PD-associated mutations trap it in a persistently activated state by impairing GTPase activity, it undergoes nucleotide-dependent conformational changes that is impaired by disease-associated mutations. The proposed studies seek to understand how these conformational changes in the ROC domain regulate the overall structure and activity of LRRK2. The three aims are: Aim 1: To determine the structures of LRRK2 and its conformational dynamics. a) Determine the structural changes in LRRK2 caused by ROC in different nucleotide-bound states and those carrying PD-associated mutations using cryo-EM. Our preliminary single-particle data reveals 2 conformations which may represent the ‘on’ and ‘off’ states. We will define these structures in the proposed studies. b) Define the atomic structures and interactions of LRRK2 by using X-ray crystallography, which is ideal for defining atoms and bonds. Conditions yielding diffracting crystals of full-length LRRK2 have been identified. c) Define the dynamic interactions of residue R1398 in ROC with the γ-phosphate of GTP using NMR, which is key in regulating mechanism of LRRK2 activity. Aim 2: To define the activities of LRRK2. a) To define the regulation of GTPase activity using measuring GTPase activity of constructs carrying PD- mutations, phosphor-memetic, and engineered conformation-constraining disulfide bonds. b) To determine the kinase active and inactive conformation of LRRK2 by measuring kinase activity of conformation-stabilized constructs. c) To characterize the activity and localization of different conformations of LRRK2 in cell models. Aim 3: To define the conformation-activity relationship of LRRK2. a) To modulate LRRK2 conformation using chemical means by employing high throughput chemical screening and chemical biology methods. b) To engineer biologics and potential therapeutic antibodies for trapping the conformations of LRRK2 by using phage-display screening methods.