Lakämper Stefan, Meyhöfer Edgar
Department of Mechanical Engineering, University of Michigan, 2350 Hayward, Ann Arbor, MI 48109, USA.
Biophys J. 2005 Nov;89(5):3223-34. doi: 10.1529/biophysj.104.057505. Epub 2005 Aug 12.
Kinesins are dimeric motor proteins that move processively along microtubules. It has been proposed that the processivity of conventional kinesins is increased by electrostatic interactions between the positively charged neck of the motor and the negatively charged C-terminus of tubulin (E-hook). In this report we challenge this anchoring hypothesis by studying the motility of a fast fungal kinesin from Neurospora crassa (NcKin). NcKin is highly processive despite lacking the positive charges in the neck. We present a detailed analysis of how proteolytic removal of the E-hook affects truncated monomeric and dimeric constructs of NcKin. Upon digestion we observe a strong reduction of the processivity and speed of dimeric motor constructs. Monomeric motors with truncated or no neck display the same reduction of microtubule gliding speed as dimeric constructs, suggesting that the E-hook interacts with the head only. The E-hook has no effect on the strongly bound states of NcKin as microtubule digestion does not alter the stall forces produced by single dimeric motors, suggesting that the E-hook affects the interaction site of the kinesin.ADP-head and the microtubule. In fact, kinetic and binding experiments indicate that removal of the E-hook shifts the binding equilibrium of the weakly attached kinesin.ADP-head toward a more strongly bound state, which may explain reduced processivity and speed on digested microtubules.
驱动蛋白是一种二聚体运动蛋白，可沿微管进行持续性移动。有人提出，传统驱动蛋白的持续性是通过马达蛋白带正电荷的颈部与微管蛋白带负电荷的C末端之间的静电相互作用（E钩）来增强的。在本报告中，我们通过研究来自粗糙脉孢菌的一种快速真菌驱动蛋白（NcKin）的运动性对这一锚定假说提出了质疑。尽管NcKin的颈部缺乏正电荷，但它具有高度的持续性。我们对蛋白酶解去除E钩如何影响NcKin的截短单体和二聚体构建体进行了详细分析。消化后，我们观察到二聚体运动构建体的持续性和速度大幅降低。截短或无颈部的单体马达与二聚体构建体表现出相同程度的微管滑动速度降低，这表明E钩仅与头部相互作用。E钩对NcKin的强结合状态没有影响，因为微管消化不会改变单个二聚体马达产生的失速力，这表明E钩影响驱动蛋白 - ADP头部与微管的相互作用位点。事实上，动力学和结合实验表明，去除E钩会使弱附着的驱动蛋白 - ADP头部的结合平衡向更强结合状态移动，这可能解释了在消化后的微管上持续性和速度降低的原因。
