Molecular Mechanism of energy transduction of dynein revealed by genetic engineering, structural and enzyme kinetics studies

本项目的主要目的是通过基因工程、结构研究和ATP酶动力学分析，阐明细胞质动力蛋白能量传递的分子机制。我们的主要结果是：（1）确定了胞质动力蛋白的最小运动域，并通过电镜观察了其结构。(2)根据ATP酶动力学，我们在最小运动域（380kDa）中鉴定了三个ATP酶位点和一个ADP结合位点，其中一个是主要的ATP酶位点，其余的是调节位点。(3)我们已经确定了ATP酶循环的中间步骤，通过停流动力学。(4)我们已经发现，从核心环突出的接头在主要ATP酶位点的ATP酶循环的特定步骤中作为臂摆动。(5)我们已经表明，微管结合结构域的动力蛋白的结构变化是在特定的ATP酶步骤的主要ATP酶网站。基于这些发现，我们提出了细胞质动力蛋白能量转导的分子模型。

Our major goal of this project is to elucidate the molecular mechanism of energy transduction of cytoplasmic dynein by genetic engineering, structural studies and ATPase kinetics analyses. Our major results are: (1) we have identified the minimal motor domain of cytoplasmic dynein and examined its structure by electron microscopy. (2) Based on ATPase kinetics, we have identified three ATPase sites and one ADP-binding site in the minimal motor domain (380kDa), among which one is the primary ATPase site and the others are regulatory sites. (3) We have identified intermediate steps of the ATPase cycle by means of stopped-flow kinetics. (4) We have discovered that the linker protruding from the core ring swings as a lever-arm at specific steps of the ATPase cycle of the primary ATPase site. (5) We have shown that structural changes of the microtubule-binding domain of dynein are induced at specific ATPase steps of the primary ATPase site. Based on these findings, we have proposed a molecular model of energy transduction of cytoplasmic dynein.