Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle
Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle
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DOI：
10.1021/bi026051l
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发表时间：
2003-01-14
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：
Sasaki, N;Ohkura, R;Sutoh, K
在网骨藻肌球蛋白11马达结构域的ATP水解循环期间，两个保守的α-螺旋，SH 1/SH 2螺旋和中继螺旋以协调的方式旋转以诱导转换器结构域的摆动运动。在这两个螺旋和转化器中的疏水和离子相互作用的网络可以确保这些螺旋的运动被有效地传递到转化器。为了研究这些相互作用在ATP酶依赖性转换器摆动中的作用，我们通过点突变（I499 A或F692 A）破坏了它们之间的两个保守疏水键。所产生的突变只引起有限的ATP水解的动力学参数的变化，除了基础MgATP酶活性的显着增加。然而，突变肌球蛋白完全失去了它们在体外和体内的运动功能。内源性色氨酸荧光和基于GFP的FRET的测量显示，这些突变体的转换器结构域在稳态ATP水解期间或在紧密捕获的Mg·ADP·V-1的存在下不摆动，这表明点突变诱导转换器摆动和ATP水解循环的解偶联。这些结果突出了这些疏水连接的重要性，用于将螺旋的协调扭转运动传输到转换器，以及该转换器摆动产生力的要求。
During the ATP hydrolysis cycle of the Dictyostelium myosin 11 motor domain, two conserved alpha-helices, the SH1/SH2 helix and the relay helix, rotate in a coordinated way to induce the swing motion of the converter domain. A network of hydrophobic and ionic interactions in these two helices and the converter may ensure that the motions of these helices are effectively transmitted to the converter. To examine the roles of these interactions in the ATPase-dependent converter swing, we disrupted two conserved hydrophobic linkages among them by means of a point mutation (I499A or F692A). The resulting mutations induced only limited changes in the kinetic parameters of ATP hydrolysis, except for a marked increase of basal MgATPase activity. However, the mutant myosins completely lost their in vitro and in vivo motor functions. Measurements of the intrinsic tryptophan fluorescence and the GFP-based FRET revealed that the converter domain of these mutants did not swing during steady-state ATP hydrolysis or in the presence of tightly trapped Mg.ADP.V-i, which shows that the point mutations induced the uncoupling of the converter swing and ATP hydrolysis cycle. These results highlight the importance of these hydrophobic linkages for transmitting the coordinated twist motions of the helices to the converter as well as the requirement of this converter swing for force generation.