Liu Jun, Wendt Thomas, Taylor Dianne, Taylor Kenneth
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA.
J Mol Biol. 2003 Jun 20;329(5):963-72. doi: 10.1016/s0022-2836(03)00516-3.
The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.
平滑肌肌球蛋白和重酶解肌球蛋白（smHMM）的肌动蛋白激活ATP酶活性受调节轻链（RLC）磷酸化的调控。完全调控需要两个完整的肌球蛋白头部，因为单头肌球蛋白亚片段总是有活性的。在带正电荷的脂质单层上制备了具有去磷酸化RLC的完整肌球蛋白10S形式的二维晶体阵列，并通过对冷冻水合标本进行低温电子显微镜以2.0 nm分辨率进行三维成像。根据平滑肌肌球蛋白运动结构域和必需轻链的X射线结构构建了平滑肌肌球蛋白的原子模型，并基于骨骼肌S1结构生成了RLC的同源模型。基于之前对smHMM的重建，对10S肌球蛋白的初始模型进行了实空间精修，以获得与密度的定量拟合。同样对smHMM进行了精修，两个精修模型都揭示了之前观察到的“受阻”头部的上部50 kDa结构域与“自由”头部的催化、转换器结构域和必需轻链部分之间相同的不对称相互作用。这一观察结果表明，这种相互作用不仅仅是由于晶体堆积，而是由肌球蛋白头部的元件所加强的。10S重建显示出在早期smHMM重建中未见到的额外α-螺旋卷曲螺旋，但两者中S2的一个片段的位置相同。
