CYTOPLASMIC DYNEIN STRUCTURE & FUNCTION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。动力蛋白运动域由六个AAA结构域和一个功能未知的C-末端结构域组成，环上有一个突出的微管结合柄。为了了解构象信息是如何在这个复杂的结构中交流的，我们生产了一系列重组的和蛋白质分解的大鼠运动区片段，并对其进行了酶学分析。令人惊讶的是，重组的210 kDa半运动结构域片段的稳态ATPase活性是380 kDa完整运动结构域片段的6倍。ATPase活性的增加与钒酸盐抑制完全丧失敏感性和ADP释放速率增加约100倍有关。产物释放的时间过程是双相的，每个阶段都被微管结合到380 kDa的运动区刺激了约1000倍。半运动结构域片段和全运动结构域片段都对胰酶蛋白分解具有显著的抵抗力，表现出两个或三个主要的切割位点。380 kDa运动区C末端附近的裂解释放了一个32 kDa的片段，并消除了对钒酸的敏感性。该位点的切割对ATP或5‘-腺基-{β}，{伽马}-亚胺二磷酸不敏感，但可被ADP-AlF3或ADP-钒酸盐阻断。基于这些数据，我们提出了一个远程变构控制AAA1和AAA3产物释放的模型，该模型通过微管结合的茎和C-末端结构域，后者可能与AAA1相互作用，以跨桥周期依赖的方式关闭运动结构域环。

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.