Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.
Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.
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DOI：
10.7554/elife.03680
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发表时间：
2014-09-10
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;Farabella I;Yu IM;Rosenfeld SS;Houdusse A;Topf M;Moores CA
动蛋白是一个基于微管的ATP驱动马达的超家族，对多种基本的细胞功能非常重要。微管结合如何刺激它们的ATPase并控制力的产生还不清楚。为了解决这个根本问题，我们使用冷冻电子显微镜在∼7？分辨率下，在ATPase周期的多个步骤中可视化了微管结合的Kinesin-1和Kinesin-3运动域-包括它们的无核苷酸状态。在这两个马达中，微管结合促进保守环的有序构象，刺激ADP释放，增强微管亲和力，并为ATP结合的催化部位准备好。ATP结合只会导致这些核苷酸协调环的小位移，但会在其他地方诱导大的构象变化，从而允许力的产生和颈部连接物对接到微管+端。运动蛋白-微管界面上的家族特异性差异解释了每个马达的不同特性。因此，我们的数据为运动蛋白的一种保守的ATP驱动机制提供了证据，并揭示了微管界面的关键机制贡献。DOI：http://dx.doi.org/10.7554/eLife.03680.001细胞内部是一个活跃的蜂巢，充满了蛋白质和其他物质从一个位置移动到另一个位置。一种被称为微管的细丝网络形成了轨道，所谓的马达蛋白沿着这些轨道携带这些物质。运动蛋白是一组运动蛋白，典型的运动蛋白有一个末端(称为‘运动结构域’)，可以附着在微管上。另一端与被运载的货物相连，两者之间有一个“脖子”连接。当这些蛋白质中的两个一起工作时，颈部的灵活区域允许两个运动域相互移动，这使得运动蛋白基本上能够以一种循序渐进的方式沿着微管行走。为了沿着微管采取这些步骤，运动域中的每个运动必须经历紧密联系和从轨迹释放的交替循环。这个循环是通过结合和分解一种名为ATP的分子来协调的，这种分子也提供了采取下一步所需的能量。松散和紧密的微管附着的循环如何与ATP分解产物的释放相协调，以及来自ATP分子的能量如何转化为沿微管移动马达的力，目前尚不清楚。阿瑟顿等人。使用一种名为冷冻电子显微镜的技术，比以前看到的更详细地研究两种类型的运动蛋白，称为运动蛋白-1和运动蛋白-3的运动域的结构。图像是在运动区域用来从ATP分子中提取能量的周期的不同阶段拍摄的。尽管这两种激动素被认为以不同的方式沿着微管轨迹移动，Atherton等人还是这样认为。发现他们的运动域使用的核心机制是相同的。当运动域与微管结合时，它的形状会改变，首先刺激前一个周期的ATP分解产物的释放。这一释放为一种新的ATP分子提供了结合的空间。ATP结合引起的结构变化相对较小，但在灵活的颈部区域产生较大的变化，使Kinesin对中的各个运动域能够协调它们的运动并朝着一致的方向移动。这一机制涉及轨道捆绑和燃料使用之间的紧密耦合，并使Kinesin成为高效电机。阿瑟顿等人发现的结构。揭示了一种将微管结合、提供给运动域的能量和沿微管移动运动蛋白的力联系起来的机制。未来的工作将阐明在运动域中观察到的运动蛋白-1和运动蛋白-3的关键特征是否也在其他类型的运动蛋白运动中被发现。DOI：http://dx.doi.org/10.7554/eLife.03680.002
Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. DOI: http://dx.doi.org/10.7554/eLife.03680.001 The interior of a cell is a hive of activity, filled with proteins and other items moving from one location to another. A network of filaments called microtubules forms tracks along which so-called motor proteins carry these items. Kinesins are one group of motor proteins, and a typical kinesin protein has one end (called the ‘motor domain’) that can attach itself to the microtubules. The other end links to the cargo being carried, and a ‘neck’ connects the two. When two of these proteins work together, flexible regions of the neck allow the two motor domains to move past one another, which enable the kinesin to essentially walk along a microtubule in a stepwise manner. To take these steps along microtubules, each kinesin motor domain in the pair must undergo alternating cycles of tight association and release from their tracks. This cycle is coordinated by binding and breaking down a molecule called ATP, which also provides the energy needed to take the next step. How the cycle of loose and tight microtubule attachment is coordinated with the release of the breakdown products of ATP, and how the energy from the ATP molecule is converted into the force that moves the motor along the microtubule, has been unclear. Atherton et al. use a technique called cryo-electron microscopy to study—in more detail than previously seen—the structure of the motor domains of two types of kinesin called kinesin-1 and kinesin-3. Images were taken at different stages of the cycle used by the motor domains to extract the energy from ATP molecules. Although the two kinesins have been thought to move along the microtubule tracks in different ways, Atherton et al. find that the core mechanism used by their motor domains is the same. When a motor domain binds to the microtubule, its shape changes, first stimulating release of the breakdown products of ATP from the previous cycle. This release makes room for a new ATP molecule to bind. The structural changes caused by ATP binding are relatively small but produce larger changes in the flexible neck region that enable individual motor domains within a kinesin pair to co-ordinate their movement and move in a consistent direction. This mechanism involves tight coupling between track binding and fuel usage and makes kinesins highly efficient motors. The structures uncovered by Atherton et al. reveal a mechanism that links microtubule binding, the energy supplied to the motor domain and the force that moves the kinesin along a microtubule. Future work will clarify whether the key features observed in the motor domains of kinesin-1 and kinesin-3 are also found in other types of kinesin motors. DOI: http://dx.doi.org/10.7554/eLife.03680.002