Deciphering the mechanisms underlying the operational plasticity of AAA+ motors

AAA+（与各种细胞活动相关的ATP酶）马达是在所有生命王国中发现的强大的生物分子机器，其将ATP结合和水解偶联以驱动各种细胞底物的机械移位的构象变化。它们在许多细胞过程中至关重要，包括蛋白质降解、蛋白质解聚和DNA复制。因此，人们对了解AAA+运动功能的分子基础非常感兴趣。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，已经提出了一个“递-递-递”模型，其中六聚体AAA+马达中的顺序ATP水解导致大体积孔环沿着底物和单向底物易位的运动。相比之下，概率模型假设，个别亚基水解ATP异步，和ATP水解的顺序不限于一个定义的顺序。这些竞争模型说明了从静态蛋白质结构推断高度动态系统（如AAA+马达）的生化机制的局限性，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如何与它们的功能相关联，并揭示和表征它们与调节和底物分子的相互作用。在这里，我专注于HslUV进化枝（HslUV，ClpAB-D2，Lon和RuvB家族）的代表性成员，高度保守的Lon和HslUV AAA+蛋白酶，其高分辨率结构可用，但仍存在许多悬而未决的问题。该提案描述了使用核磁共振（NMR）光谱和氢氘交换质谱（HDX-MS）的溶液的组合的Lon和HslUV蛋白酶的综合结构和动力学表征。这两种方法，再加上生化技术，提供了独特的机械见解AAA+电机的功能。我提出了以下短期和中期的具体目标：（1）理解AAA+马达中协同性的作用;（2）表征与Lon和HslUV结合的底物;（3）表征AAA+蛋白质中的结构动力学和功能变构。通过我的组合NMR-MS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EM结构，并有望提供AAA+马达和机械化学酶在细胞中的功能的详细视图。这三个精心整合的目标，结合我的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。

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.