Shimada T, Sasaki N, Ohkura R, Sutoh K
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Tokyo 153, Japan.
Biochemistry. 1997 Nov 18;36(46):14037-43. doi: 10.1021/bi971837i.
In order to determine the functional roles of the conserved sequence (NXNSSRFG) of the "switch I" loop (residues 233-240 in Dictyostelium myosin II), alanine scanning mutagenesis was performed on Dictyostelium myosin II. N233A and S237A mutant myosins did not bind a fluorescent analog of ADP, mant-deoxyADP, at the low concentration range (micromolar and had low level of ATPase activities. They were nonmotile when examined by the in vitro motility assay. Dictyostelium cells expressing these myosins showed worse phenotypes than that of myosin-null cells. In contrast to these mutant myosins, R238A myosin tightly bound mant-deoxyADP. However, the mutant had a defect in the ATP hydrolysis step and exhibited the lowest ATPase activities among the mutants examined here. The R238A myosin was nonmotile. R238C or R238H mutations, which mimic the Usher syndrome mutations, generated myosins with similar functional defects to those of the R238A mutation. Cells expressing the R238A myosin exhibited the phenotype similar to that of the myosin-null cells. N235A, S236A, F239A, and G240A myosins retained moderate levels of ATPase activities and could drive sliding of actin filaments at various speeds. Phenotypes of cells expressing them were very similar to that of the wild-type cells. Taken together, these results suggest that side chains of N233 and S237 may play essential roles in holding a nucleotide in the ATPase pocket and that R238 may play crucial roles in the ATP hydrolysis step, while those of the other residues in the switch I loop are not essential for the process.
为了确定“开关I”环（盘基网柄菌肌球蛋白II中第233 - 240位氨基酸残基）保守序列（NXNSSRFG）的功能作用，对盘基网柄菌肌球蛋白II进行了丙氨酸扫描诱变。N233A和S237A突变型肌球蛋白在低浓度范围（微摩尔）下不结合ADP的荧光类似物mant - 脱氧ADP，并且ATP酶活性水平较低。通过体外运动分析检测时它们不具有运动能力。表达这些肌球蛋白的盘基网柄菌细胞表现出比肌球蛋白缺失细胞更差的表型。与这些突变型肌球蛋白相反，R238A肌球蛋白紧密结合mant - 脱氧ADP。然而，该突变体在ATP水解步骤存在缺陷，并且在此处检测的突变体中表现出最低的ATP酶活性。R238A肌球蛋白不具有运动能力。模拟乌舍尔综合征突变的R238C或R238H突变产生了具有与R238A突变类似功能缺陷的肌球蛋白。表达R238A肌球蛋白的细胞表现出与肌球蛋白缺失细胞类似的表型。N235A、S236A、F239A和G240A肌球蛋白保留了中等水平的ATP酶活性，并且能够以不同速度驱动肌动蛋白丝的滑动。表达它们的细胞的表型与野生型细胞非常相似。综上所述，这些结果表明N233和S237的侧链可能在将核苷酸保持在ATP酶口袋中起重要作用，而R238可能在ATP水解步骤中起关键作用，而开关I环中其他残基的侧链对于该过程并非必不可少。
