WPI, Immunology Frontier Research Center, Osaka University, Osaka, Japan.
Biochemistry. 2010 Jun 8;49(22):4654-61. doi: 10.1021/bi9021582.
Kinesin-1 is an ATP-driven molecular motor that transports various cargoes in cells, a process that can be regulated by the kinesin tail domain. Here, kinesin ATPase activity and motility were inhibited in vitro by interacting the kinesin heavy chain C-terminal tail domain with the kinesin N-terminal motor domain. Though the tail domain can directly interact with microtubules, we found 70% of tail domains failed to bind in the presence of >100 mM (high) KCl, which also modulated the ATPase inhibition manner. These observations suggest that self-inhibition of kinesin depends on electrostatic interactions between the motor domain, the tail domain, and a microtubule. Furthermore, we observed self-regulated behavior of kinesin at the single molecule level. The tail domain did not affect motility velocity, but it did lower the binding affinity of the motor domain to the microtubule. The decrement in binding was coupled to ATPase inhibition. Meanwhile, the tail domain transfected into living cells not only failed to bind to microtubules but also inhibited the motor domain and microtubule interaction, in agreement with our in vitro results. Furthermore, at high potassium concentrations, the self-regulation of kinesin observed in cells was like that in vitro. The results favor a way tail inhibition mechanism where the tail domain masks the microtubule binding site of the motor domain in high potassium concentration.
驱动蛋白-1 是一种 ATP 驱动的分子马达，能够在细胞内运输各种货物，这一过程可以通过驱动蛋白尾部结构域来调节。在这里，通过将驱动蛋白重链 C 末端尾部结构域与驱动蛋白 N 末端马达结构域相互作用，在体外抑制了驱动蛋白的 ATP 酶活性和运动。虽然尾部结构域可以直接与微管相互作用，但我们发现 70%的尾部结构域在存在>100mM（高）KCl 的情况下无法结合，这也调节了 ATP 酶抑制方式。这些观察结果表明，驱动蛋白的自我抑制取决于马达结构域、尾部结构域和微管之间的静电相互作用。此外，我们在单分子水平上观察到了驱动蛋白的自我调控行为。尾部结构域不影响运动速度，但会降低马达结构域与微管的结合亲和力。结合的减少与 ATP 酶抑制有关。同时，转染到活细胞中的尾部结构域不仅不能与微管结合，还抑制了马达结构域与微管的相互作用，与我们的体外结果一致。此外，在高钾浓度下，细胞内观察到的驱动蛋白的自我调节与体外相似。这些结果支持了一种尾部抑制机制，即尾部结构域在高钾浓度下掩盖了马达结构域的微管结合位点。
