Zhang Wen-Bo, Yao Lin-Lin, Li Xiang-Dong
From the Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China and the University of Chinese Academy of Sciences, Beijing 100049, China.
From the Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China and.
J Biol Chem. 2016 Jun 24;291(26):13571-9. doi: 10.1074/jbc.M116.724328. Epub 2016 Apr 28.
Myosin-5a contains two heavy chains, which are dimerized via the coiled-coil regions. Thus, myosin-5a comprises two heads and two globular tail domains (GTDs). The GTD is the inhibitory domain that binds to the head and inhibits its motor function. Although the two-headed structure is essential for the processive movement of myosin-5a along actin filaments, little is known about the role of GTD dimerization. Here, we investigated the effect of GTD dimerization on its inhibitory activity. We found that the potent inhibitory activity of the GTD is dependent on its dimerization by the preceding coiled-coil regions, indicating synergistic interactions between the two GTDs and the two heads of myosin-5a. Moreover, we found that alanine mutations of the two conserved basic residues at N-terminal extension of the GTD not only weaken the inhibitory activity of the GTD but also enhance the activation of myosin-5a by its cargo-binding protein melanophilin (Mlph). These results are consistent with the GTD forming a head to head dimer, in which the N-terminal extension of the GTD interacts with the Mlph-binding site in the counterpart GTD. The Mlph-binding site at the GTD-GTD interface must be exposed prior to the binding of Mlph. We therefore propose that the inhibited Myo5a is equilibrated between the folded state, in which the Mlph-binding site is buried, and the preactivated state, in which the Mlph-binding site is exposed, and that Mlph is able to bind to the Myo5a in preactivated state and activates its motor function.
肌球蛋白-5a包含两条重链，它们通过卷曲螺旋区域形成二聚体。因此，肌球蛋白-5a由两个头部和两个球状尾部结构域（GTD）组成。GTD是与头部结合并抑制其运动功能的抑制结构域。尽管双头结构对于肌球蛋白-5a沿肌动蛋白丝的持续运动至关重要，但关于GTD二聚化的作用知之甚少。在这里，我们研究了GTD二聚化对其抑制活性的影响。我们发现GTD的有效抑制活性取决于其通过前面的卷曲螺旋区域形成的二聚体，这表明两个GTD与肌球蛋白-5a的两个头部之间存在协同相互作用。此外，我们发现GTD N端延伸处两个保守碱性残基的丙氨酸突变不仅削弱了GTD的抑制活性，还增强了其货物结合蛋白亲黑素（Mlph）对肌球蛋白-5a的激活作用。这些结果与GTD形成头对头二聚体一致，其中GTD的N端延伸与对应GTD中的Mlph结合位点相互作用。在Mlph结合之前，GTD-GTD界面处的Mlph结合位点必须暴露。因此，我们提出，被抑制的Myo5a在折叠状态（其中Mlph结合位点被掩埋）和预激活状态（其中Mlph结合位点暴露）之间达到平衡，并且Mlph能够结合处于预激活状态的Myo5a并激活其运动功能。
