Klumpp Lisa M, Mackey Andrew T, Farrell Christopher M, Rosenberg John M, Gilbert Susan P
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
J Biol Chem. 2003 Oct 3;278(40):39059-67. doi: 10.1074/jbc.M304250200. Epub 2003 Jul 14.
Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.
开关I和开关II是驱动蛋白、肌球蛋白和G蛋白关键的活性位点结构元件。我们对果蝇驱动蛋白中开关I突变体（R210A）的分析表明，其微管亲和力降低，运动结构域之间的协同性丧失，且存在ATP水解缺陷，导致从微管上异常脱离。为了进一步研究开关I中保守的精氨酸，我们构建了一个赖氨酸替代突变体。R210K二聚体运动蛋白失去了水解ATP的能力；然而，它挽救了微管功能。我们的结果表明，R210K恢复了微管结合动力学、微管亲和力、ADP释放动力学以及运动结构域的协同性。此外，头部1的活性位点能够区分ATP、ADP和AMP-PNP，从而向头部2发出信号使其结合微管并释放mantADP，其动力学与野生型相当。因此，从头部1到头部2的结构通讯途径得以恢复，头部2能够通过结合微管并释放mantADP来响应这一信号。结构建模显示，赖氨酸能够保留精氨酸形成的部分氢键，但并非全部，这为赖氨酸在Arg210突变体中挽救微管功能的能力提出了一个结构假说。
