Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains
Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains
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DOI：
10.1073/pnas.1417422112
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发表时间：
2015-05-19
影响因子：
11.1
通讯作者：
Gennerich, Arne
中科院分区：
文献类型：
--
作者：
Nicholas, Matthew P.;Berger, Florian;Gennerich, Arne
胞浆动力蛋白是一种同源二聚体微管运动蛋白，负责大部分微管末端运动。动力蛋白的每个运动域(AAA1-4)包含四个AAA+ATPase(AAA：与各种细胞活动相关的ATPase)。大多数动力蛋白动力学模型认为，ATP的主要水解点AAA1是唯一被考虑的位点。然而，目前还不清楚ATPase活性和MT结合是如何在动力蛋白的运动域内部和之间协调的。利用光学镊子，我们表征了重组动力蛋白单体的MT结合强度作为机械张力和核苷酸状态的函数。动力蛋白对张力的反应是各向异性的，当拉向MT+端时，它与MTS结合得更紧密。我们提供的证据表明，这种行为是由于一种不对称的粘结造成的，这种粘结在前向张力下起滑移粘结作用，在后向张力下起滑移理想粘结作用。ATP削弱了MT的结合，降低了键强度的各向异性，ADP也是如此。使用核苷酸结合和水解突变体，我们表明，虽然ATP通过结合AAA1发挥作用，但ADP的作用是由AAA3介导的。最后，我们通过AAA3演示了AAA1函数的“门控”功能。当没有张力或通过动力蛋白的C末端施加张力时，ATP与AAA1的结合仅在AAA3处于水后分解状态时才诱导MT释放。然而，当张力施加到连接子上时，与AAA3结合的ATP足以打开门。这些结果阐明了动力蛋白-MT相互作用的机制，确定了AAA3的调节作用，并有助于确定机械张力和核苷酸状态在调节动力蛋白运动中的相互作用。
Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.