Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
J Biol Chem. 2011 Jun 24;286(25):22300-7. doi: 10.1074/jbc.M111.219014. Epub 2011 May 2.
The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.
肌球蛋白利用 ATP 水解产生的能量产生力和运动。重要的保守组件（P 环、开关 I 和开关 II）有助于将活性位点的小构象变化传播到蛋白质远端区域的大构象变化。结构和生化研究表明，开关 I 可能直接负责在 ATP 酶循环中肌动球蛋白和核苷酸结合口袋的相互打开和关闭，从而有助于协调这些重要的底物结合位点。平滑肌肌球蛋白已经显示出能够同时紧密结合肌动蛋白和 ADP 的能力，尽管如果它们与开关 I 刚性耦合，不清楚如何关闭两个底物结合裂隙。在这里，我们使用平滑肌肌球蛋白的单个色氨酸突变体来确定在肌动蛋白和 ADP 存在下，开关 I 中的构象变化如何与核苷酸和肌动蛋白结合裂隙的结构变化相关联。我们的结果表明，在强结合的肌球蛋白-ADP 复合物中，封闭的开关 I 构象负责维持紧密的核苷酸结合，尽管核苷酸结合口袋是打开的。这种独特的状态对于平滑肌中长时间的张力维持可能是至关重要的。
