Allosteric Role of Dynein Light Chains in Dynein Assembly and Regulation

细胞质动力蛋白是一种以微管为基础的分子马达，它利用三磷酸腺苷水解产生的能量将细胞内的物质移动和运输到微管的负端。这种运输在细胞行为的几个方面是必不可少的，包括细胞迁移和分裂、染色体分离和囊泡运输。动力蛋白重链亚基与微管结合，包含提供动力的ATP位点，而轻链和中间链被认为与货物结合，调节马达的活动。Barbar实验室的最新数据表明，轻链LC8的作用是促进动力蛋白中间链天然无序的N-末端结构域的二聚化，而不是通常认为的主要功能是作为货物适配器。轻链Tctex-1是Lc8的结构同系物，在与Lc8结合位点相邻的位置结合中间链，很可能具有与Lc8相似的作用。该研究项目将检验轻链Lc8和Tctex-1的串联作用是促进dynein组装和与货物适配器dynactin相互作用的假设，而不是作为货物适配器本身。Barbar实验室将结合包括核磁共振(核磁共振)、等温滴定热法和X射线结晶学在内的生物物理方法，阐明两个轻链与动力蛋白中间链相互作用的三个重要结果的机制：1)它们如何有助于形成紧密的动力蛋白组装，2)它们如何促进中间链的二聚化，以及3)它们如何促进中间链与动力蛋白的p150Glued亚单位更紧密的相互作用。这些轻链的基本作用将在该项目的合作者汤姆·海斯教授的实验室中进行活体测试。这些研究将为动力蛋白运动功能的结构生物学和热力学提供新的见解，并将补充在细胞内运输领域占主导地位的细胞生物学方法。由于这些研究首次提供了关于无序到有序转变在动力蛋白组装中的作用及其与动力蛋白的关联的结构见解，它们使得有机会开发一个易于处理的系统来探索内在无序在大分子复合体组装中的作用。更广泛的影响：PI在本科生的研究贡献以及指导少数族裔和残疾学生方面有着良好的记录。国际和平研究所在她的校园里发挥着领导作用，通过提供进入仪器和培训研讨会的途径，将分子生物物理学介绍给研究界。此外，PI将通过提供研究实验室体验的机会，并通过在星期六学院计划中开发教学材料和动手模块，在高中生中发挥积极作用，促进基础科学。后者是俄勒冈州立大学一个非常成功的项目，面向还没有参加过生物化学和生物物理学课程的高中生。

Cytoplasmic dynein is a microtubule-based molecular motor that uses the energy derived from ATP hydrolysis to move and transport cellular cargo towards the minus ends of microtubules. This transport is essential in several aspects of cell behavior including cell migration and division, chromosome segregation, and vesicle trafficking. The dynein heavy chain subunits bind to microtubules and contain the ATP sites that provide the motive force, while the light and intermediate chains are thought to bind to cargo and regulate the activity of the motor. Recent data from the Barbar lab have suggested that the role of light chain LC8 is to facilitate dimerization of the natively disordered N-terminal domain of dynein intermediate chain, rather than to primarily function as a cargo adaptor, as commonly thought. Light chain Tctex-1, which is a structural homolog of LC8 and binds the intermediate chain at a site contiguous to the LC8 binding site, is likely to have a similar role as LC8. The research project will test the hypothesis that the tandem roles of light chains LC8 and Tctex-1 are to promote dynein assembly and the interaction with the cargo adaptor dynactin, and not to act as cargo adaptors themselves. Using a combination of biophysical approaches including nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry and X-ray crystallography, the Barbar lab will elucidate the mechanisms of three important outcomes of the interactions of both light chains to dynein intermediate chain: 1) how they contribute to formation of a tight dynein assembly, 2) how they promote dimerization of the intermediate chain, and 3) how they may promote a tighter interaction of the intermediate chain with p150Glued subunit of dynactin. The essential roles of these light chains will be tested in vivo in the lab of Professor Tom Hays, a collaborator on this project. The studies should provide novel insights into the structural biology and thermodynamics of dynein motor function, and will complement the cell biological approaches that predominate in the field of intracellular transport. Since these studies provide the first structural insights into the role of the disorder-to-order transition in dynein assembly and its association with dynactin, they allow the opportunity to develop a tractable system to probe the role of intrinsic disorder in the assembly of large macromolecular complexes. Broader Impacts: The PI has a strong record of research contributions with undergraduate students, and of mentoring minorities and students with disabilities. The PI is taking a leading role on her campus to introduce molecular biophysics to the research community by providing access to instrumentation and training workshops. Furthermore, the PI will take an active role to promote basic science among high school students by providing opportunities for research lab experience, and by developing instructional material and a hands-on module in the Saturday Academy program. The latter is a highly successful program at Oregon State University for outreach for high school students in which biochemistry and biophysics have not yet been represented.