Alanine scanning mutagenesis of the switch I region in the ATPase site of Dictyostelium discoideum myosin II
Alanine scanning mutagenesis of the switch I region in the ATPase site of Dictyostelium discoideum myosin II
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DOI：
10.1021/bi971837i
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发表时间：
1997-11-18
期刊：
影响因子：
2.9
通讯作者：
Sutoh, K
中科院分区：
文献类型：
--
作者：
Shimada, T;Sasaki, N;Sutoh, K
为了确定‘’Switch I‘’环的保守序列(Dictyostelialmyosin II中的233-240残基)的功能作用，对Dictyostelialmyosin II进行了丙氨酸扫描突变。N233A和S237A突变的肌球蛋白在低浓度(微摩尔)范围内不与ADP的荧光类似物mant-deoxADP结合，并具有低水平的ATPase活性。经体外运动试验检测，均为非运动性。表达这些肌球蛋白的Dictyostelialcell表现出比肌球蛋白缺失的细胞更差的表型。与这些突变的肌球蛋白相比，R238A肌球蛋白与mant-deoxADP紧密结合。然而，该突变体在ATP水解步骤中存在缺陷，并且在所检测的突变体中显示出最低的ATPase活性。R238A肌球蛋白是不运动的。模拟Usher综合征突变的R238C或R238H突变产生的肌球蛋白具有与R238A突变相似的功能缺陷。表达R238A肌球蛋白的细胞表现出与肌球蛋白缺失细胞相似的表型。N235A、S236A、F239A和G240A肌球蛋白保持中等水平的ATPase活性，并能以不同的速度驱动肌动蛋白细丝的滑动。表达它们的细胞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ATPase口袋中起关键作用，R238可能在ATP水解步骤中发挥关键作用，而Switch I环中其他残基的侧链对这一过程不是必需的。
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.