The Mechanism of Kinesin Self-Regulation

截短的驱动蛋白二聚体水解ATP并沿沿着单向移动的机制 微管是很好理解的。目前还不清楚完整的驱动蛋白异四聚体，其中有两个重链， 链和两条轻链，被调节和激活用于货物运输。在这项工作中，我们将测试 假设当尾部直接结合头部以阻止ADP释放时，驱动蛋白受到调节， 微管结合驱动蛋白可以通过其轻链和重链之间的电荷冲突来进一步调节。 当磷酸化的轻链竞争微管的尾部时，驱动蛋白可以被重新激活， 头。我们将在四个具体目标中检验这些假设。前两个目标使用 全长驱动蛋白重链，后两个目的是探索轻链在调节中的作用， 和活动。在目标#1中，我们将确定尾是否直接结合在微管结合位点或与微管结合位点结合。 头部的核苷酸敏感元件，或者它是否会变构影响核苷酸-或 头部的微管结合区。目标#2的实验，在目标#1的结果的指导下，将 确定头部的哪个区域与尾部结合，并将识别特定的头部-尾部相互作用。实验 在体内和体外进行的研究表明，轻链可能在调节 驱动蛋白，将在目标#3中评估。最后，我们将确定驱动蛋白的磷酸化是否 链可以直接激活目标#4中的驱动蛋白。总之，这些实验将扩展我们对 控制驱动蛋白活性的相互作用和构象变化。此外，监管 在这项工作中发现的相互作用可能揭示了在几种驱动蛋白中类似的抑制机制。 这可能会导致更快地发现专门针对驱动蛋白的药物。

The mechanism by which truncated kinesin dimers hydrolyze ATP and moves unidirectionally along microtubules is well understood. It is far less clear how the full kinesin heterotetramer, which has two heavy chains and two light chains, is regulated and activated for cargo transport. In this work, we will test the hypothesis that kinesin is regulated when the tails directly bind the heads to prevent ADP release or microtubule binding. Kinesin may be further regulated by a charge clash between its light chains and microtubules, and kinesin may be re-activated when phosphorylated light chains compete the tails away from the heads. We will test these hypotheses in four Specific Aims. The first two Aims address regulation using the full-length kinesin heavy chain, and the second two Aims explore the role of the light chains in regulation and activiation. In Aim #1, we will determine whether the tail binds directly in the microtuble-binding site or to the nucleotide-sensing elements in the head, or whether it allosterically affects the nucleotide- or microtubule-binding regions of the head. The experiments of Aim #2, guided by the results of Aim #1, will determine what region of the head binds the tail and will identify specific head-tail interactions. Experiments performed both in vivo and in vitro indicate that the light chains may have a significant role in regulating kinesin, which will be assessed in Aim #3. Lastly, we will determine whether phosphorylation of kinesin light chains can directly activate kinesin in Aim #4. Together, these experiments will extend our understanding of the interactions and conformational changes that govern kinesin activity. Furthermore, the regulatory interactions that are found in this work may reveal inhibitory mechanisms that are similar in several kinesins. This may lead to quicker discovery of drugs that specifically target kinesins.