Examining kinesin processivity within a general gating framework.
Examining kinesin processivity within a general gating framework.
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DOI：
10.7554/elife.07403
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发表时间：
2015-04-22
期刊：
影响因子：
7.7
通讯作者：
Block SM
中科院分区：
文献类型：
--
作者：
Andreasson JO;Milic B;Chen GY;Guydosh NR;Hancock WO;Block SM
驱动蛋白-1是一种二聚体马达，其沿着沿着微管运输货物，以双手交替的方式进行8.2纳米的步骤。通过一系列的门控机制，其两个头部的ATP水解循环保持异相，这导致平均<1.1 μm的连续运行。头间协调的一个关键结构元件是颈连接器（NL），它将头连接到茎。为了研究NL在调节步进中的作用，我们在门控的一般框架的背景下，使用单分子光捕获和批量荧光方法研究了各种长度的NL突变体。我们的研究结果表明，虽然头间张力提高电机速度，它是至关重要的头间协调，也不快速后脑释放。此外，半胱氨酸轻突变体在负荷下不产生野生型运动性。我们的结论是，驱动蛋白-1主要是前端门控，和NL的长度调整，以提高单向的持续合成能力和速度。http://dx.doi.org/10.7554/eLife.07403.001在细胞中，分子通过马达蛋白从一个位置移动到另一个位置。驱动蛋白是这样的马达的一个大家族，其沿着被称为微管的长细丝运输其货物。大多数驱动蛋白分子由两条相同的蛋白质链形成。每条链的一端（称为头部）都有一个运动区，可以附着在微管上。每条蛋白质链的另一端缠绕在它的伙伴周围，形成一个共同的茎区（称为尾巴），连接到所携带的货物。两个驱动蛋白的头部通过一个“颈部连接器”区域连接到尾部，它们沿着微管严格交替前进，类似于我们走路时腿的运动方式。在每一步中，前头部保持与细丝紧密相连，而后头部释放自己，向前头部前进，并重新连接成为新的前头部。这两个头需要协调他们的活动，这样在任何给定的时间，他们都不会处于过程的同一阶段。例如，如果两个头部同时与微管结合，马达将无法前进。如果它们都释放，电机就会从灯丝上脱落并扩散。然而，头部协调的过程还没有完全理解，并且已经提出了这个过程如何工作的不同模型。现在，Andreasson，Milic等人已经研究了颈部连接器在协调运动中所起的作用，使用一种称为“光学陷阱”的技术。这些实验涉及将微观珠子附着在马达蛋白上，作为可以跟踪的标记。这些珠子也可以用来对驱动蛋白分子施加受控的力，以观察它们如何对不同的负载做出反应。Andreasson，Milic等人通过在蛋白质的这个区域插入额外的氨基酸（其是蛋白质的构建单元）来延长颈接头的长度。研究发现，即使每个颈连接体被延长多达6个额外的氨基酸，驱动蛋白仍然可以行走。然而，即使是在接头中引入一个氨基酸，也会放松头部与细丝结合时头部之间存在的正常张力。这导致速度减慢，行程缩短，承受载荷的能力降低。实验结果表明，天然存在的驱动蛋白中颈连接体的长度可以进行优化以支持最大运动。根据他们的数据，Andreasson，Milic等人提出了一个通用框架，用于理解头部之间需要进行的通信，以便以协调的方式行走。需要进一步的工作，以了解除了驱动蛋白以外的马达蛋白是否也可以用相同的框架来理解。DOI：http://dx.doi.org/10.7554/eLife.07403.002网站
Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity. DOI: http://dx.doi.org/10.7554/eLife.07403.001 In cells, molecules are moved from one location to another by motor proteins. Kinesins are a large family of such motors that transport their cargos along long filaments known as microtubules. Most kinesin molecules are formed from two identical protein chains. Each chain has a motor region at one end (called the head) that can attach to microtubules. The other end of each protein chain wraps around its partner to form a common stalk region (called the tail) that links to the cargo being carried. The two kinesin heads are connected to the tail via a ‘neck linker’ region, and they advance along the microtubule in strict alternation, similar to the way our legs move when walking. During each step, the front head remains tightly associated with the filament as the trailing head releases itself, advances beyond the front head, and reattaches to become the new leading head. The two heads need to coordinate their activities, so that at any given time, they're not at the same stage in the process. For example, if both heads remained bound to the microtubule at the same time, the motor would not be able to advance. If they both released, the motor would fall off the filament and diffuse away. However, the process by which the heads coordinate is not fully understood, and different models for how this process works have been proposed. Now, Andreasson, Milic et al. have examined the role played by the neck linker in coordinating the motor's movement using a technique known as ‘optical trapping’. The experiments involved attaching microscopic beads to the motor proteins, which serve as markers that can be tracked. The beads can also be used to exert controlled forces on the kinesin molecules, to see how they respond to different loads. Andreasson, Milic et al. extended the length of neck linker by inserting extra amino acids (which are the building blocks of proteins) into this region of the protein. It was found that kinesins can still walk even when each neck linker was extended by up to six additional amino acids. However, introducing even a single amino acid into the linker relaxed the normal tension that exists between the heads when these are both bound to the filament. This resulted in slowed speeds, shorter distances of travel, and less ability to sustain loads. The experimental results suggest that the length of the neck linker in naturally occurring kinesins may be optimized to support maximum movement. Based on their data, Andreasson, Milic et al. propose a general framework for understanding the communication that needs to take place between the heads in order to walk in a coordinated manner. Further work is required to understand if motor proteins other than kinesins can also be understood with this same framework. DOI: http://dx.doi.org/10.7554/eLife.07403.002