Ajtai Katalin, Halstead Miriam F, Nyitrai Miklós, Penheiter Alan R, Zheng Ye, Burghardt Thomas P
Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Biochemistry. 2009 Jun 16;48(23):5263-75. doi: 10.1021/bi900584q.
Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V(max). In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop.
肌动蛋白和肌球蛋白构成了肌肉中的分子马达。肌球蛋白是一种在肌动蛋白的变构控制下进行ATP水解的酶，使得肌动蛋白结合引发产物释放以及肌球蛋白动力冲程中的力产生。动力冲程的非平衡蒙特卡罗分子动力学模拟表明，肌球蛋白上的一个结构化表面环——C环，是启动肌球蛋白ATP酶肌动蛋白激活的肌动蛋白接触传感器。先前的实验工作表明C环与肌动蛋白结合，并建立了C环与肌球蛋白活性位点之间的正向和反向变构联系。在此，构建了平滑肌重酶解肌球蛋白C环嵌合体，用骨骼肌（sCl）和心肌（cCl）肌球蛋白C环取代天然序列。在这两种情况下，肌动蛋白激活的ATP酶抑制主要表现为较低的V(max)。嵌合体的体外运动性也受到抑制。收集了野生型、cCl和sCl在不同肌球蛋白表面密度、有无调节的肌动蛋白以及与原肌球蛋白结合的骨骼肌和心肌同工型肌动蛋白条件下的运动数据。在野生型物种中发现了肌球蛋白速度的慢和快亚群，分别代表几何上不利和有利的肌动球蛋白相互作用。在所有测试的表面密度下都检测到了不利相互作用。在较高的肌球蛋白表面密度下，有利相互作用更有可能发生。与心肌原肌球蛋白结合的肌动蛋白通过降低对肌动蛋白的结构效应来降低抑制性几何约束障碍，从而促进有利的肌动球蛋白相互作用。更高的表面密度和与心肌原肌球蛋白结合的肌动蛋白都不能加速cCl或sCl中的运动速度，这表明启动肌动蛋白对肌球蛋白最大激活的元件位于C环中。
