Wendt T, Taylor D, Trybus K M, Taylor K
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4361-6. doi: 10.1073/pnas.071051098. Epub 2001 Apr 3.
Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically "blocked" because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.
已知平滑肌肌球蛋白II的肌动蛋白激活ATP酶的调节涉及两个头部之间的相互作用，该相互作用由调节轻链的磷酸化控制。然而，这种失活形式的三维结构一直未知。我们利用脂质单层获得了平滑肌重酶解肌球蛋白未磷酸化失活形式的二维晶体阵列，适用于通过对未染色、冷冻水合标本进行电子冷冻显微镜结构研究。三维结构揭示了两个肌球蛋白头部之间的不对称相互作用。一个头部的ATP酶活性在空间上被“阻断”，因为其肌动蛋白结合界面的一部分位于第二个头部的转换器结构域上。第二个能够结合肌动蛋白的头部的ATP酶活性，似乎是通过稳定释放磷酸和实现强肌动蛋白结合所需的转换器结构域运动而受到抑制。当重酶解肌球蛋白的亚片段2结构域按其在肌动球蛋白丝晶格中的方向排列时，头部的位置与结合肌动蛋白所需的位置非常不同，这表明对原位ATP酶抑制有额外贡献。
