Structural characterization of single, double and triple-headed axonemal dyneins

单头、双头和三头轴丝动力蛋白的结构表征

• 批准号: 10518523
• 负责人: Alan Brown
• 金额: $ 37.72万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10518523
• 关键词: ATP phosphohydrolase; Affect; Animal Model; Biochemical Genetics; Bos taurus; Calcium; Cattle; Cerebrospinal Fluid; Chemicals; Chlamydomonas; Chlamydomonas reinhardtii; Chronic; Cilia; Cilium Microtubule; Complex; Cryoelectron Microscopy; Data; Diagnosis; Docking; Dynein ATPase; Elements; Embryonic Development; Etiology; Evolution; Extracellular Fluid; Family; Fertility; Flagella; Frequencies; Functional disorder; Genetic study; Handedness; Head; Hereditary Disease; Human; Infertility; Lead; Location; Locomotion; Mechanics; Methods; Microtubules; Molecular; Motor; Movement; Mucous body substance; Mutagenesis; Mutation; Natural Immunity; Neonatal Respiratory Distress; Nucleotides; Oocytes; Organelles; Organism; Paralysed; Pattern; Periodicity; Physiology; Play; Positioning Attribute; Radial; Regulation; Resolution; Respiratory Tract Infections; Role; Speed; Structure; Video Microscopy; Work; arm; base; cell motility; chronic respiratory disease; ciliopathy; cilium motility; driving force; fluid flow; insight; new therapeutic target; particle; preservation; respiratory; response; sperm cell; targeted treatment

Project Summary Motile cilia play essential roles in fertility, innate immunity, and embryonic development. The beat of motile cilia is powered by a diverse family of ATP-dependent motors called axonemal dyneins. Axonemal dyneins are tethered in repeating patterns to doublet microtubules within the ciliary axoneme and are classified by the number of motor domains (or heads) that they contain and their position within the axoneme. Outer dynein arms (ODA) are either double or triple-headed complexes that repeat every 24 nm, whereas inner dynein arms (IDA) are either double or single-headed and repeat every 96 nm. Their different positions, periodicities, and subunit compositions manifest as different activities: the ODA determines the beat frequency, whereas the IDA determines the amplitude of the waveform. Despite their fundamental importance to ciliary motility and human physiology, little is known about the structures and mechanisms of the large axonemal dynein family. In this proposal, we plan to exploit recent advances in single-particle electron cryomicroscopy (cryo-EM) to determine structures of all major classes of axonemal dynein. To capture axonemal dyneins in their active, microtubule- bound states we have developed methods to isolate and determine high-resolution structures of native dynein- bound doublet microtubules from three organisms - the biflagellate model organism Chlamydomonas reinhardtii, Bos taurus and humans. C. reinhardtii will be used for structural, biochemical, and genetic studies of a triple-headed ODA (Aim 1) and double and single-headed IDAs (Aim 2). These structures are expected to reveal the mechanisms that dock axonemal dyneins to the doublet microtubule, the structural rearrangements they undergo during the powerstroke, their regulation by calcium and microtubule curvature, and the functional relevance of inter-dynein interactions in generating the ciliary waveform. Structures of single and multi-headed axonemal dyneins will provide insights into the functions of their idiosyncratic subunits and the general principles that have guided evolution of the axonemal dynein family. Structures of dynein-bound doublet microtubules from humans and cows (Aim 3) will reveal their differences with algal axonemal dyneins (for example the comparison between double and triple-headed ODAs) and help explain the etiology of ciliopathy- causing mutations. Mutations in axonemal dyneins are the leading cause of primary ciliary dyskinesis (PCD), a currently incurable inherited disease characterized by neonatal respiratory distress, chronic airway infections, and infertility. Advances in understanding the structures of axonemal dyneins will therefore have important implications for the identification of key mechanisms that can targeted for therapy of defective cilia.

项目概要 运动纤毛在生育力、先天免疫和胚胎发育中发挥着重要作用。运动纤毛的节拍 由称为轴丝动力蛋白的多种 ATP 依赖性马达家族提供动力。轴丝动力蛋白是 以重复模式束缚在睫状轴丝内的双联微管上，并按 它们包含的运动域（或头）的数量及其在轴丝中的位置。外动力蛋白 臂 (ODA) 是每 24 nm 重复一次的双头或三头复合物，而内部动力蛋白臂 (IDA) 为双头或单头，每 96 nm 重复一次。它们的位置、周期不同， 亚基组成表现为不同的活性：ODA 决定拍频，而 IDA 决定拍频。 确定波形的幅度。尽管它们对纤毛运动和人类具有根本重要性 生理学方面，人们对轴丝动力蛋白大家族的结构和机制知之甚少。在这个 根据提案，我们计划利用单粒子电子冷冻显微镜 (cryo-EM) 的最新进展来确定 轴丝动力蛋白所有主要类别的结构。捕获活性微管中的轴丝动力蛋白 我们已经开发出分离和确定天然动力蛋白的高分辨率结构的方法 来自三种生物体的结合双联微管 - 双鞭毛模式生物衣藻 莱茵哈特牛、牛和人类。莱茵衣藻将用于结构、生化和遗传研究 三头 ODA（目标 1）和双头和单头 IDA（目标 2）。这些结构预计将 揭示轴丝动力蛋白与双联微管对接的机制，结构重排 它们在动力冲程期间经历钙和微管曲率的调节以及功能 动力蛋白间相互作用在产生纤毛波形中的相关性。单头和多头的结构 轴丝动力蛋白将提供对其特殊亚基和一般功能的见解 指导轴丝动力蛋白家族进化的原则。动力蛋白结合双峰的结构 来自人类和牛的微管（目标 3）将揭示它们与藻类轴丝动力蛋白的差异（例如 例如双头和三头 ODA 之间的比较）并有助于解释纤毛病的病因- 引起突变。轴丝动力蛋白突变是原发性纤毛运动障碍 (PCD) 的主要原因 目前无法治愈的遗传性疾病，其特征是新生儿呼吸窘迫、慢性气道感染、 和不孕症。因此，了解轴丝动力蛋白结构的进展将具有重要意义。 对识别可靶向治疗有缺陷纤毛的关键机制的意义。