Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein
Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein
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DOI：
10.1021/bi060991e
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发表时间：
2006-10-17
期刊：
影响因子：
2.9
通讯作者：
Nyitray, Laszlo
中科院分区：
文献类型：
--
作者：
Hodi, Zsuzsa;Nemeth, Attila L.;Nyitray, Laszlo
一个10 kDa的动力蛋白轻链(DLC)，以前被认为是肌球蛋白Va的尾部轻链，可能是肌球蛋白和动力蛋白的一个货物结合和/或调节亚基。在这里，我们鉴定和表征了DLC与肌球蛋白Va的结合部位。表达了人肌球蛋白Va尾和DLC2亚型的片段，并通过下拉分析、凝胶过滤和光谱方法分析了它们的复合体形成。DLC2被发现作为同源二聚体与一个类似于15个残基的片段(Ile1280-Ile1294)结合，该片段位于尾部的内侧和远端盘绕结构域之间。结合区含有由选择性剪接的外显子B(Asp1284-Lys1286)编码的三个残基。外显子B的去除消除了DLC2的结合。在转基因哺乳动物细胞系中的共定位实验证实了我们的发现，外显子B是DLC2结合所必需的。利用圆二色谱，我们证明了DLC2与类似于85个残基的无序结构域(Pro1235-Arg1320)结合后产生了一些螺旋结构，并稳定了两侧的螺旋线圈结构域(熔融温度上升约7℃)。这一结果表明，DLC2促进了肌球蛋白Va卷曲结构域的组装。核磁共振波谱和对接模拟表明，15个残基多肽(Ile1280-Ile1294)与DLC2上的表面沟槽结合，类似于DLC的其他已知结合伙伴。当我们的数据结合在一起时，他们表明外显子B及其相关的DLC2对部分卷曲尾区的结构有显著影响，这种方式可能会影响肌球蛋白Va的调节和货物结合功能。
A 10 kDa dynein light chain (DLC), previously identified as a tail light chain of myosin Va, may function as a cargo-binding and/or regulatory subunit of both myosin and dynein. Here, we identify and characterize the binding site of DLC on myosin Va. Fragments of the human myosin Va tail and the DLC2 isoform were expressed, and their complex formation was analyzed by pull- down assays, gel filtration, and spectroscopic methods. DLC2 was found to bind as a homodimer to a similar to 15 residue segment (Ile1280-Ile1294) localized between the medial and distal coiled-coil domains of the tail. The binding region contains the three residues coded by the alternatively spliced exon B (Asp1284-Lys1286). Removal of exon B eliminates DLC2 binding. Co-localization experiments in a transfected mammalian cell line confirm our finding that exon B is essential for DLC2 binding. Using circular dichroism, we demonstrate that binding of DLC2 to a similar to 85 residue disordered domain (Pro1235-Arg1320) induces some helical structure and stabilizes both flanking coiled-coil domains (melting temperature increases by similar to 7 degrees C). This result shows that DLC2 promotes the assembly of the coiled-coil domains of myosin Va. Nuclear magnetic resonance spectroscopy and docking simulations show that a 15 residue peptide (Ile1280-Ile1294) binds to the surface grooves on DLC2 similarly to other known binding partners of DLCs. When our data are taken together, they suggest that exon B and its associated DLC2 have a significant effect on the structure of parts of the coiled-coil tail domains and such a way could influence the regulation and cargo-binding function of myosin Va.