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驱动蛋白-3 NcKin3运动结构域的特性及其对颈部功能的影响。

Properties of the kinesin-3 NcKin3 motor domain and implications for neck function.

作者信息

Adio Sarah, Woehlke Günther

机构信息

Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

出版信息

FEBS J. 2009 Jul;276(13):3641-55. doi: 10.1111/j.1742-4658.2009.07083.x. Epub 2009 May 28.

Abstract

Members of the Kinesin-3 family are microtubule motors involved in the transport of membranous cargo. NcKin3 from the fungus Neurospora crassa is dimeric but inactivates one of its motor heads to generate nonprocessive motility. To determine how one of the heads is inactivated, we investigated truncated monomeric constructs. None of the constructs generated processive single-molecule motility, and multimotor velocities depended linearly on the number of residues remaining in the neck. The kinetic analysis suggests futile ATP hydrolysis cycles, because a representative monomer showed a faster ATP turnover than the dimer while supporting slower motility. The K(0.5,MT) was 70-fold lower, the microtubule-bound portion of the kinetic cycle eight-fold longer and the microtubule detachment rate almost 15-fold slower than that of the dimer. Moreover, the monomer's microtubule-dependent ADP release occurred three-fold to four-fold faster than k(cat) (125 versus 34 s(-1)), whereas phosphate release was approximately equally fast (29 s(-1)). A dimeric construct containing a structure-breaking insert between motor head and neck showed a similar behaviour. These data suggest that the heads of the wild-type NcKin3 motor are strictly coupled via the neck domain, and that the dimeric structure is required for proper detachment after one ATPase cycle. This is the first direct comparison of a monomeric Kinesin-3 with its dimeric full-length counterpart, and the kinetic changes observed here may also apply to other Kinesin-3 motors.

摘要

驱动蛋白-3家族成员是参与膜性货物运输的微管马达。来自粗糙脉孢菌的NcKin3是二聚体,但会使其一个马达头部失活以产生非持续性运动。为了确定其中一个头部是如何失活的,我们研究了截短的单体构建体。没有一个构建体产生持续性单分子运动,多马达速度与颈部剩余残基数量呈线性相关。动力学分析表明存在无效的ATP水解循环,因为一个代表性单体在支持较慢运动的同时,其ATP周转速度比二聚体更快。单体的K(0.5,MT)比二聚体低70倍,动力学循环中与微管结合的部分比二聚体长8倍,微管脱离速率比二聚体慢近15倍。此外,单体的微管依赖性ADP释放比k(cat)快三到四倍(125对34 s(-1)),而磷酸盐释放速度大致相同(29 s(-1))。一个在马达头部和颈部之间含有破坏结构插入物的二聚体构建体表现出类似的行为。这些数据表明,野生型NcKin3马达的头部通过颈部结构域严格耦合,并且二聚体结构是一个ATP酶循环后正确脱离所必需的。这是首次将单体驱动蛋白-3与其二聚体全长对应物进行直接比较,此处观察到的动力学变化可能也适用于其他驱动蛋白-3马达。

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