The affinity of the dynein microtubule-binding domain is modulated by the conformation of its coiled-coil stalk
The affinity of the dynein microtubule-binding domain is modulated by the conformation of its coiled-coil stalk
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DOI：
10.1074/jbc.m501636200
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发表时间：
2005-06-24
影响因子：
4.8
通讯作者：
Carter, AP
中科院分区：
文献类型：
--
作者：
Gibbons, IR;Garbarino, JE;Carter, AP
动力蛋白的微管结合域 (MTBD) 通过类似 15 nm 的茎与马达的 AAA（具有任何其他活性的 ATP 酶）核心分开，该茎预计由反向平行卷曲线圈组成。然而，这种卷曲线圈的结构及其用于介导 MTBD 和 ATP 结合核心之间通信的机制尚不清楚。在这里，我们试图确定茎卷曲线圈两条链中疏水七肽重复序列之间的最佳对齐方式。为此，我们将小鼠细胞质动力蛋白的 MTBD 与其茎的 12-36 个残基一起融合到嗜热栖热菌丝氨酰-tRNA 合成酶提供的稳定卷曲螺旋碱基上，并在体外测试这些嵌合构建体的微管结合。结果确定了一种比对，该比对产生了对微管表现出高亲和力的蛋白质（2.2 μM）。应用于该构建体的 MTBD 的突变的影响与先前报道的在没有 ATP 的情况下完整动力蛋白运动单元的影响相似（Koonce, M. P., and Tikhonenko, I. (2000) Mol. Biol. Cell 11, 523-529），表明它类似于天然完整动力蛋白的紧密结合状态。所有其他比对显示对微管的亲和力至少低 10 倍，但其中一个除外，其具有中等亲和力。基于这些结果和氨基酸序列分析，我们假设动力蛋白利用其卷曲螺旋茎的两条链之间的少量滑动位移作为机械化学循环期间马达的 AAA 核心和 MTBD 之间的通讯手段。
The microtubule-binding domain ( MTBD) of dynein is separated from the AAA ( ATPase with any other activity) core of the motor by an similar to 15-nm stalk that is predicted to consist of an antiparallel coiled coil. However, the structure of this coiled coil and the mechanism it uses to mediate communication between the MTBD and ATP-binding core are unknown. Here, we sought to identify the optimal alignment between the hydrophobic heptad repeats in the two strands of the stalk coiled coil. To do this, we fused the MTBD of mouse cytoplasmic dynein, together with 12-36 residues of its stalk, onto a stable coiled-coil base provided by Thermus thermophilus seryl-tRNA synthetase and tested these chimeric constructs for microtubule binding in vitro. The results identified one alignment that yielded a protein displaying high affinity for microtubules ( 2.2 mu M). The effects of mutations applied to the MTBD of this construct paralleled those previously reported (Koonce, M. P., and Tikhonenko, I. (2000) Mol. Biol. Cell 11, 523-529) for an intact dynein motor unit in the absence of ATP, suggesting that it resembles the tight binding state of native intact dynein. All other alignments showed at least 10-fold lower affinity for microtubules with the exception of one, which had an intermediate affinity. Based on these results and on amino acid sequence analysis, we hypothesize that dynein utilizes small amounts of sliding displacement between the two strands of its coiled-coil stalk as a means of communication between the AAA core of the motor and the MTBD during the mechanochemical cycle.