Deciphering the mechanisms underlying the operational plasticity of AAA+ motors

破译 AAA 电机运行可塑性的机制

• 批准号: RGPIN-2021-02843
• 负责人: Vahidi, Siavash
• 金额: $ 2.99万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761168
• 关键词: Deciphering; mechanisms; underlying; operational; plasticity

AAA+ (ATPases associated with various cellular activities) motors are powerful biomolecular machines found in all kingdoms of life that couple ATP binding and hydrolysis to drive conformational changes for the mechanical translocation of a variety of cellular substrates. They are critical in many cellular processes including protein degradation, protein disaggregation, and DNA replication. As such, there is significant interest in understanding the molecular basis of AAA+ motor function. Despite numerous high-resolution structures over the past decade, this has proven to be a formidable challenge. Based on these structures, a `hand-over-hand' model has been proposed where sequential ATP hydrolysis in hexameric AAA+ motors results in movement of bulky pore loops along the substrate and unidirectional substrate translocation. By contrast, the probabilistic model posits that individual subunits hydrolyze ATP asynchronously, and the sequence of ATP hydrolysis is not restricted to a defined order. These competing models illustrate the limits of inferring biochemical mechanisms of highly dynamic systems, such as AAA+ motors, from static protein structures and demonstrate the need for the application of complementary methodologies capable of probing structural plasticity. The long-term goals of my research program are to understand how the structure and dynamics of all seven evolutionary clades of AAA+ motors are linked to their function, and to uncover and characterize their interactions with regulatory and substrate molecules. Here I focus on representative members of the HCLR clade (HslUV, ClpAB-D2, Lon, and RuvB family), the highly conserved Lon and HslUV AAA+ proteases, for which high-resolution structures are available yet many outstanding questions remain. This proposal describes a comprehensive structural and dynamical characterization of the Lon and HslUV proteases using a combination of solution on nuclear magnetic resonance (NMR) spectroscopy and hydrogen deuterium exchange mass spectrometry (HDX-MS). These two methods, together with biochemical techniques, provide unique mechanistic insights into the function of AAA+ motors. I propose the following short- and medium-term specific aims: (1) Understanding the role of cooperativity in AAA+ motors; (2) Characterization of substrate binding to Lon and HslUV; and (3) Characterization of structural dynamics and functional allostery in AAA+ proteins. Insights into protein dynamics obtained via my combined NMR-MS approach complement existing X-ray and cryo-EM structures and promise to provide a detailed view of the function of AAA+ motors and mechanochemical enzymes in cells. These three carefully integrated aims, combined with my HQP Training Plan that focuses on flexible mentorship to accommodate different HQP learning styles, needs, and backgrounds, guarantees a fertile ground for training the next generation of scientists.

AAA+（与各种细胞活动相关的ATP酶）马达是一种强大的生物分子机器，存在于所有生物王国中，它将ATP结合和水解结合起来，驱动各种细胞底物的机械易位的构象变化。它们在许多细胞过程中起关键作用，包括蛋白质降解、蛋白质分解和DNA复制。因此，了解AAA+运动功能的分子基础具有重要意义。尽管在过去十年中有许多高分辨率结构，但这已被证明是一项艰巨的挑战。基于这些结构，提出了一个“手把手”模型，其中六聚体AAA+马达中的顺序ATP水解导致沿底物移动的大孔环和单向底物易位。相比之下，概率模型假设单个亚基不同步水解ATP，并且ATP水解的顺序不受限制。这些相互竞争的模型说明了从静态蛋白质结构推断高动态系统（如AAA+马达）生化机制的局限性，并证明了应用能够探测结构可塑性的互补方法的必要性。我的研究计划的长期目标是了解AAA+马达的所有七个进化分支的结构和动力学是如何与其功能联系在一起的，并揭示和表征它们与调节和底物分子的相互作用。在这里，我重点关注HCLR分支的代表成员（HslUV, clab - d2， Lon和RuvB家族），高度保守的Lon和HslUV AAA+蛋白酶，它们的高分辨率结构是可用的，但仍有许多悬而未决的问题。本文采用核磁共振（NMR）光谱和氢氘交换质谱（HDX-MS）相结合的方法对Lon和hslv蛋白酶进行了全面的结构和动力学表征。这两种方法与生化技术一起，为AAA+电机的功能提供了独特的机械见解。我提出以下中短期具体目标：(1)理解协同在AAA+电机中的作用；(2)底物与Lon和HslUV结合的表征；(3) AAA+蛋白的结构动力学和功能变构表征。通过我的联合核磁共振-质谱方法获得的蛋白质动力学见解补充了现有的x射线和冷冻电镜结构，并有望提供细胞中AAA+马达和机械化学酶功能的详细视图。这三个精心整合的目标，加上我的HQP培训计划，重点是灵活的指导，以适应不同的HQP学习风格、需求和背景，保证了培养下一代科学家的肥沃土壤。