The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.
The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.
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DOI：
10.7554/elife.27793
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发表时间：
2017-08-11
期刊：
影响因子：
7.7
通讯作者：
Moores CA
中科院分区：
文献类型：
--
作者：
Atherton J;Yu IM;Cook A;Muretta JM;Joseph A;Major J;Sourigues Y;Clause J;Topf M;Rosenfeld SS;Houdusse A;Moores CA
MKLP2 是一种驱动蛋白 6，在中期-后期转变和胞质分裂过程中发挥着关键作用。其运动结构域包含保守的核苷酸结合基序，但与其他驱动蛋白相比，其序列（约 35% 同一性）和大小（约大 40%）不同。使用冷冻电子显微镜和生物物理测定，我们对 ATP 酶循环期间微管结合的 MKLP2 运动结构域进行了机械化学解剖，并表明其机制的许多方面与其他驱动蛋白不同。虽然 MKLP2 颈连接子以类似 ATP 的状态指向微管正端，但它并不完全沿着运动域对接。此外，与运动驱动蛋白相比，MKLP2 运动结构域在 MT 表面上的足迹发生了改变，并通过驱动蛋白 6 特异性序列得到增强。驱动蛋白 6 的高度延伸的 Loop6 插入特征的构象是不依赖于核苷酸的，并且不接触 MT 表面。我们的结果强调了家族特异性插入在调节驱动蛋白运动功能中的作用。细胞不断复制，为生长的组织提供新细胞，并替换身体周围老化或有缺陷的细胞。每个新细胞都需要遗传物质的副本，称为有丝分裂纺锤体的细胞结构可确保当细胞一分为二时正确共享该物质。纺锤体由称为微管的蛋白质丝构成，随着有丝分裂纺锤体发挥其作用，蛋白质丝会生长和收缩。纺锤体的许多变化是由称为分子马达的蛋白质驱动的，分子马达会分解富含能量的 ATP 分子，为它们沿着细丝行走提供动力。例如，驱动蛋白是可以沿着微管移动的分子马达，人类基因组中编码了 40 多种不同的驱动蛋白。超过一半的人类驱动蛋白参与细胞分裂，其中包括一种名为 MKLP2 的驱动蛋白。人们对 MKLP2 知之甚少，但一些早期的发现表明，与其他驱动蛋白相比，它的行为有很大不同。了解驱动蛋白马达的工作原理需要对其微管轨道进行复杂的研究。阿瑟顿，余等人。现在，他们使用了一种称为冷冻电子显微镜的技术，该技术特别适合在三维空间中观察大型且复杂的样品，以观察 MKLP2 中的电机在工作时如何改变形状。这表明，虽然 MKLP2 的工作方式与其他驱动蛋白基本相似，但其分子机制的许多方面都非常不寻常。其中包括它如何与微管结合、如何与 ATP 相互作用以及如何产生力。这些发现表明，参与细胞分裂的驱动蛋白的分子机制比以前认为的要多样化得多。几种抗癌药物靶向驱动蛋白来阻止细胞分裂，因此这种多样性可能使药物更容易仅靶向某些驱动蛋白，从而减少副作用。不过，首先，重要的是要找出 MKLP2 的不寻常机制如何协调和影响纺锤体的其他组成部分，以更全面地揭示细胞复制时发生的情况。
MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function. Cells constantly replicate to provide new cells for growing tissues, and to replace ageing or defective cells around the body. Each new cell needs a copy of the genetic material, and a cellular structure called the mitotic spindle makes sure that this material is shared correctly when a cell divides in two. The spindle is built from protein filaments called microtubules, and the protein filaments grow and shrink as the mitotic spindle carries out its role. Many of these changes in the spindle are driven by proteins called molecular motors, which break down energy-rich molecules of ATP to power them as they walk along the filaments. Kinesins, for example, are molecular motors that can move along microtubules and there are over 40 different kinesins encoded in the human genome. More than half of the human kinesins are involved in cell division including one called MKLP2. Little is known about MKLP2 but some earlier findings had suggested that it would behave very differently compared to other kinesins. Understanding how a kinesin motor works requires studying it in complex with its microtubule tracks. Atherton, Yu et al. have now used a technique called cryo-electron microscopy – which is uniquely suited to looking at large and complicated samples in three dimensions – to observe how the motor in MKLP2 changes shape as it works. This revealed that, while MKLP2 works in a fundamentally similar way to other kinesins, many aspects of its molecular mechanism are highly unusual. These include how it binds to the microtubule, how it interacts with ATP and how it generates force. These findings show that there is much greater diversity in the molecular mechanisms of the kinesins involved in cell division than was previously thought. Several anticancer drugs target kinesins to stop cells dividing and so this diversity may make it easier to target only certain kinesins with drugs, which in turn would have fewer side effects. First, though, it will be important to find out how the unusual mechanism of MKLP2 coordinates and influences other components of the spindle to reveal a fuller picture of what happens when cells replicate.