Studies on Molecular Mechanism of Force Generation by Recombinant Dynein

我们已经成功地在网骨藻细胞中表达了网骨藻细胞质动力蛋白的重组片段（Nishiura等人，J.Biol.Chem（2004））。表达的分子量为380 kDa的动力蛋白片段由C-末端重链片段组成，并保持完整的马达活性：该片段是一个单头动力蛋白马达，具有高的微管激活ATP酶活性（120 s^<-1>），并以3 μm/sec的速度驱动微管滑动。通过微管着陆试验，滑动被证明是非进行性的，占空比被确定为0.37。通过组合这些值，预期步长为8 nm。因此，这种活性重组细胞质动力蛋白片段的行为类似于单头驱动蛋白，另一种基于微管的马达。通过使用这种独特的表达系统，我们已经研究了动力蛋白运动域的AAA+环中的四个潜在的ATP酶位点的功能作用。我们在这四个潜在的ATP酶位点的高度保守的步行者A和步行者B序列中引入了点突变，并检查了它们的基础和微管激活的ATP酶活性以及它们的运动性。我们已经发现，第一个AAA+模块具有负责力产生的主要ATP酶位点，并且第三个AAA+模块具有与前者紧密偶联的ATP酶位点（Kon等人，Biochemistry（2004））。然后，我们开发了基于GFP的FRET系统来检测动力蛋白的ATP水解周期依赖性结构转变，其可能对应于动力冲程（Kon et al.，Nature Structural and Molecular Biology（2005））。我们确定了两个不同的结构状态，具有不同的荧光光谱，由于荧光共振能量转移连接到两个位置上的活性动力蛋白马达的GFP和BFP之间。我们还将这两种状态与动力蛋白初级ATP酶位点的环状ATP水解的中间状态相关联。

We have succeeded to express recombinant fragment of Dictyostelium cytoplasmic dynein in Dictyostelium cells (Nishiura et al. J.Biol.Chem (2004)). The expressed dynein fragment with molecular mass of 380kDa is consisted of the C-terminal heavy chain fragment, and maintains full motor activities : the fragment, a single-headed dynein motor, has high microtubule-activated ATPase activity (120 s^<-1>), and drives microtubule sliding at 3 μm/sec. By microtubule-landing assays, the sliding is shown to be non-processive, and the duty ratio is determined as 0.37. By combining these values, the step size is expected to be 8 nm. Thus, this active recombinant cytoplasmic dynein fragment behaves like the single-headed kinesin, another microtubule-based motor. By using this unique expression system, we have examined functional roles of four potential ATPase sites in the AAA+-ring of the dynein motor domain. We introduced point mutations in the highly conserved Walker A and Walker B sequences in these four potential ATPase sites, and examined their basal and microtubule-activated ATPase activities as well as their motility. We have found that the first AAA+ module has the primary ATPase site responsible for the force generation, and the third AAA+ module has the ATPase site tightly coupled with the former (Kon et al., Biochemistry (2004)). We have then developed the GFP-based FRET system to detect ATP hydrolysis cycle-dependent structural transitions of dynein, which may correspond to power stroke (Kon et al., Nature Structural and Molecular Biology (2005)). We identified two distinct structural states that have distinct fluorescence spectra due to FRET between GFP and BFP attached to two locations on the active dynein motor. We also correlated these two states to intermediate states of cyclic ATP hydrolysis at the dynein primary ATPase site.