Clark Richard, Ansari Maqsood Ali, Dash Sheffali, Geeves Michael A, Coluccio Lynne M
Department of Biosciences, University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, United Kingdom.
J Biol Chem. 2005 Sep 2;280(35):30935-42. doi: 10.1074/jbc.M504698200. Epub 2005 Jun 26.
Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.
环1是肌球蛋白运动结构域中的一个柔性表面环，部分构成了位于核苷酸结合位点附近的转换区域。根据结构研究推测，该区域负责ATP水解后产物释放的动力学调节（1）。生化研究表明，环1可能通过无机磷酸（Pi）的释放影响肌动蛋白-肌球蛋白-II对ADP的亲和力、运动性以及肌动蛋白激活的Mg2+-ATP酶活性的最大反应速度（Vmax）（2 - 8）。为了测试环1对哺乳动物I类肌球蛋白Myo1b的影响，构建了嵌合分子，其中：（i）将截短形式的Myo1b即Myo1b1IQ的环1替换为其他肌球蛋白的环1；（ii）将环1替换为甘氨酸；或（iii）将环1中的一些氨基酸替换为丙氨酸，并在杆状病毒中进行表达，然后评估它们与肌动蛋白和核苷酸的相互作用。与野生型相比，嵌合体的稳态肌动蛋白激活的ATP酶活性、ATP诱导的肌动蛋白从Myo1b1IQ解离的速率、ADP从肌动蛋白-Myo1b1IQ释放的速率以及肌动蛋白对Myo1b1IQ和Myo1b1IQ-ADP的亲和力均有所不同，这表明环1对肌动蛋白与核苷酸结合事件之间偶联的影响范围比之前认为的要广泛得多。特别是，ATP诱导的肌动蛋白从肌动蛋白-Myo1b1IQ的双相解离在嵌合体中发生了显著改变。这提供了证据表明环1有助于核苷酸口袋的可及性，并参与来自肌动蛋白、核苷酸、γ-Pi和钙调蛋白结合位点信息的整合，同时预测环1调节了马达的负载依赖性。
