High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation.
High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation.
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DOI：
10.7554/elife.04686
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发表时间：
2014-11-21
期刊：
影响因子：
7.7
通讯作者：
Sindelar CV
中科院分区：
文献类型：
--
作者：
Shang Z;Zhou K;Xu C;Csencsits R;Cochran JC;Sindelar CV
由ATP水解提供动力的驱动蛋白马达基于微管的运输对于真核生物中广泛的重要过程是必不可少的。我们获得了深入了解这一过程，通过开发原子模型的无核苷酸和ATP状态的单体驱动蛋白运动域的微管从cryo-EM重建在5 - 6 μ m的分辨率。通过比较这些模型与现有的ADP结合的驱动蛋白的X射线结构，我们推断一个机制的方案，其中微管连接，介导的驱动蛋白（N255）中的一个普遍保守的“关键”残基，触发一个蛤壳打开的核苷酸裂缝和伴随的ADP释放。ATP的结合通过驱动蛋白的"颈连接体"元件以与货物的易位紧密耦合的方式重新闭合裂缝。这些结构转变让人想起肌球蛋白和F1-ATP酶马达中类似的核苷酸交换步骤，并告知驱动蛋白二聚体的两个头部如何彼此"门控"以促进沿着沿着微管的协调步进。细胞内部是一个动态的环境。蛋白质等大分子通常通过"马达蛋白"在细胞内运输，马达蛋白沿着沿着称为微管的细丝网络移动。一组马达蛋白，驱动蛋白，通常有一个被称为马达结构域的末端，它附着在微管上。另一端连接到被携带的货物上，一个灵活的“颈部”区域连接着马达蛋白的两端。驱动蛋白成对地结合在一起。柔性的颈部区域允许成对的每个运动域移动经过另一个，允许驱动蛋白以阶梯状的方式沿着微管"行走"沿着。每一步都需要一个运动域交替地与微管细丝紧密结合，然后从微管细丝释放。这种交替循环是由驱动蛋白结合并分解一种称为ATP的分子以形成另一种称为ADP的分子来协调的，ADP释放下一步所需的能量。这种重复的循环是可能的，因为马达结构域在与微管结合时会改变形状。这种形状的变化刺激ADP的释放，为新的ATP分子释放空间，以结合到运动域。虽然相对较小，但这些结构变化在柔性颈部区域产生较大的变化，使驱动蛋白对内的各个运动域能够协调它们的运动并有效地运动。许多先前的研究已经使用一种称为冷冻电子显微镜的技术研究了这些形状的变化，该技术可以快速冷冻样品，并允许高度详细地记录它们的结构。然而，运动域的小尺寸及其形状的变化意味着这种技术无法完全详细地揭示结构。Shang等人现在利用低温电子显微镜的最新进展来更详细地检查单个驱动蛋白运动域的结构变化。当运动域处于两种不同状态之一时，运动域与微管结合的图像被制作出来：不与ATP或ADP结合，或者与不能被分解的ATP的化学修饰形式结合。Shang等人随后使用这些图像生成运动域的模型，并将模型与先前发表的图像进行比较。这揭示了驱动蛋白运动域中的一个裂缝，当它附着在微管上时会打开。这个裂缝的"蛤壳状"开口允许ADP被释放;然后当ATP分子与它结合时，它关闭。裂缝的打开和关闭导致驱动蛋白的"颈连接器"的变化，使马达蛋白能够运输其货物，从而将ATP结合与马达蛋白的运动联系起来。Shang等人认为，类似的过程也可能发生在其他马达蛋白中，这些马达蛋白没有像驱动蛋白那样得到很好的研究。DOI：www.example.com网站
Microtubule-based transport by the kinesin motors, powered by ATP hydrolysis, is essential for a wide range of vital processes in eukaryotes. We obtained insight into this process by developing atomic models for no-nucleotide and ATP states of the monomeric kinesin motor domain on microtubules from cryo-EM reconstructions at 5–6 Å resolution. By comparing these models with existing X-ray structures of ADP-bound kinesin, we infer a mechanistic scheme in which microtubule attachment, mediated by a universally conserved ‘linchpin’ residue in kinesin (N255), triggers a clamshell opening of the nucleotide cleft and accompanying release of ADP. Binding of ATP re-closes the cleft in a manner that tightly couples to translocation of cargo, via kinesin's ‘neck linker’ element. These structural transitions are reminiscent of the analogous nucleotide-exchange steps in the myosin and F1-ATPase motors and inform how the two heads of a kinesin dimer ‘gate’ each other to promote coordinated stepping along microtubules. DOI: http://dx.doi.org/10.7554/eLife.04686.001 The inside of a cell is a dynamic environment. Large molecules such as proteins are commonly transported within a cell by ‘motor proteins’, which move along a network of filaments called microtubules. One group of motor proteins, the kinesins, typically have one end called a motor domain that attaches itself to a microtubule. The other end links to the cargo being carried, and a flexible ‘neck’ region connects the two ends of the motor protein. Kinesins are bound together in pairs. The flexible neck region allows each motor domain in a pair to move past that of the other, allowing the kinesin to ‘walk’ along a microtubule in a step-like manner. Each step requires one motor domain to alternately tightly associate with, and then release from, a microtubule filament. This alternating cycle is coordinated by kinesin binding to and breaking down a molecule called ATP to form another molecule called ADP, which releases the energy needed for its next step. This repeating cycle is possible because a motor domain changes shape when it binds to a microtubule. This shape change stimulates the release of ADP, freeing up room for a new ATP molecule to bind to the motor domain. Although relatively small, these structural changes produce larger changes in the flexible neck region that enable the individual motor domains within a kinesin pair to co-ordinate their movement and move efficiently. Many previous studies have investigated these shape changes using a technique called cryo-electron microscopy, which rapidly freezes samples and allows their structure to be recorded in high detail. However, the small size of the motor domains and their changes in shape means that this technique was not able to reveal the structures in full detail. Shang et al. now exploit recent advances in cryo-electron microscopy to examine the structural changes of individual kinesin motor domains in greater detail. Images of motor domains bound to microtubules were made while the motor domain was in one of two different states: not bound to ATP or ADP, or bound to a chemically modified form of ATP that cannot be broken down. Shang et al. then used these images to produce models of the motor domains and compared the models with previously published images. This revealed a cleft in the kinesin motor domain that opens when it attaches to a microtubule. This cleft's ‘clamshell-like’ opening allows ADP to be released; it then closes when a molecule of ATP binds to it. The opening and closing of the cleft causes the changes in the ‘neck linker’ of the kinesin that enable the motor protein to transport its cargo, and so links ATP binding to the movement of the motor protein. Shang et al. suggest that similar processes may also occur in other motor proteins that have not been as well studied as the kinesins. DOI: http://dx.doi.org/10.7554/eLife.04686.002