CYTOPLASMIC DYNEIN STRUCTURE & FUNCTION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。dynein马达结构域由6个具有突出微管结合柄的AAA结构域环和一个功能未知的c端结构域组成。为了了解构象信息是如何在这个复杂的结构中传递的，我们制作了一系列重组和蛋白水解的大鼠运动结构域片段，并对其进行酶分析。令人惊讶的是，重组的210 kda半运动结构域片段比380 kda完全运动结构域片段表现出高6倍的稳态atp酶活性。atp酶活性的增加与对钒酸盐抑制的敏感性完全丧失以及ADP释放速度增加约100倍有关。发现产物释放的时间过程是双相的，每一相都受到微管与380-kDa马达结构域结合约1000倍的刺激。半运动结构域和全运动结构域片段对色氨酸蛋白水解都有明显的抗性，表现出两个或三个主要的裂解位点。380 kda马达结构域C端附近的切割释放了32 kda片段，并消除了对钒酸盐的敏感性。该位点的切割对ATP或5'-腺苷基-{β}，{γ}-酰亚胺二磷酸不敏感，但被ADP-AlF3或adp -钒酸盐阻断。基于这些数据，我们提出了一个通过微管结合柄和c端结构域对AAA1和AAA3的产物释放进行长期变构控制的模型，后者可能与AAA1相互作用，以跨桥周期依赖的方式关闭马达结构域环。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an ~100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated ~1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5'-adenylyl-{beta},{gamma}-imidodiphosphate but was blocked by ADP-AlF3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.