CYTOPLASMIC DYNEIN STRUCTURE & FUNCTION

细胞质动力蛋白结构

• 批准号: 7355075
• 负责人: Richard Bert Vallee
• 金额: $ 0.25万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355075
• 关键词: CYTOPLASMIC; DYNEIN; STRUCTURE; FUNCTION

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。动力蛋白运动结构域由一个六个AAA结构域的环组成，具有突出的微管结合茎和一个未知功能的C末端结构域。为了了解如何在这种复杂结构中传达构象信息，我们生成了一系列的重组和蛋白水解大鼠运动结构域片段，我们通过酶法进行了分析。重组210 kDa半运动结构域片段出人意料地表现出比380 kDa完整的运动域片段高6倍的稳态ATPase活性。 ATPase活性的增加与对钒酸盐抑制的敏感性完全丧失，ADP释放速率增加了约100倍。发现产品释放的时间过程是双相的，并且通过微管结合与380 kDa运动结构域刺激了每个相约1000倍。半运动和完整的运动结构域碎片都对胰蛋白酶蛋白水解具有明显抗性，表现出两个或三个主要的切割位点。 380 kDa运动域的C末端附近的裂解释放了32 kDa片段，并消除了对糊状物的敏感性。该站点的裂解对ATP或5'- adenylyl- {beta}，{gamma} -imidodiph磷酸盐不敏感，但被ADP-ALF3或ADP-Vanadate阻止。基于这些数据，我们提出了一个模型，用于通过微管结合茎和C末端结构域在AAA1和AAA3处对产品释放的远程变构控制​​，后者可能与AAA1相互作用，以以跨桥循环依赖性方式关闭运动域环。