Molecular mechanism of protein motors revealed by the combination of genetic engineering and single-molecule analysis

我们已经构建了一个仅由重链(760个残基)组成的肌球蛋白运动域，它保留了运动功能。通过这种结构，我们成功地结晶了肌球蛋白的运动结构域，并在原子分辨率(2.7A)下求解了它的结构。利用这些结构信息，我们对肌球蛋白的重要功能区域进行了定点突变，特别是Switch I和Switch II环。由Dictyostelialmyosin II的454-459残基组成的环在结构和功能上与G蛋白家族的Switch II环相同。肌球蛋白家族Switch II环的共同序列为DIXGFE，为了确定每个保守残基的功能，对Dictyostelialmyosin II重链基因进行了丙氨酸扫描突变。对突变型肌球蛋白的体内和体外运动功能的检测表明，I455A和S456A突变体保留了这些功能，而D454A、G457A、F458A和E459A突变体失去了这些功能。对后者的生化分析表明，G457A和E459A突变体分别通过阻断ATPase循环的异构化和水解峰而失去了基础的ATPase活性。然而，F458A突变体失去了肌动蛋白激活的ATPase活性，而没有失去基础ATPase活性。根据Dictyostelialmyosin运动域的晶体结构对这些结果进行了讨论。

We have constructed a myosin motor domain consisted of the heavy chain alone(760 residues), which retained motor functions. By using this construct, we succeeded to crystallize the myosin motor domain, and solve its structure at the atomic resolution (2.7A). By using this structural information, we carried out carried out site-directed mutagenesis of functionally important regions of myosin, especially the switch I and switch II loops. A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the switch II loop of the myosin family is DIXGFE.In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions while the D454A,G457A,F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis speps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.