Ikebe M, Yamada M, Mabuchi K, Kambara T, Ikebe R
Department of Physiology, University of Massachusetts Medical Center, Worcester 01655-0127, USA.
Biochemistry. 1999 Aug 17;38(33):10768-74. doi: 10.1021/bi990915u.
A recent report has suggested that the interaction between the head and the rod region of smooth muscle myosin at S2 is important for the phosphorylation-mediated regulation of myosin motor activity [Trybus, K. M., Freyzon, Y., Faust, L. Z., and Sweeney, H. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 74, 48-52]. To investigate whether specific amino acid residues at S2 or whether the registration of the 7-residue/28-residue repeat appearing in the alpha-helical coiled-coil structure of the rod are critical for such an interaction, two smooth muscle myosin mutants were constructed in which the N-terminal sequences of S2 were deleted to various extents. One mutant contained a deletion of 71 residues at the position immediately C-terminal to the invariant proline (Pro849) linking the S1 domain directly to the downstream sequence of the rod, while in another mutant, 53 residues were deleted at a position 56 residues downstream of Pro849. Despite these alterations which change the registration of both the 28-residue repeat and the 7-residue repeat found in myosin rod sequence, both myosin mutants showed a stable double-headed structure by electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the two mutant myosins was the same as the wild-type recombinant myosin. Furthermore, the head configuration critical for myosin filament formation (extended or folded) was unchanged in either mutant. These results indicate that neither the specific amino acid residues nor the registration of the amino acid repeat in S2 is critical for the head configuration. These results indicate that neither a specific amino acid sequence at the head-rod junction nor the rod sequence registration is critical for the regulation of smooth muscle myosin.
最近的一份报告表明，平滑肌肌球蛋白在S2处头部与杆状区域之间的相互作用对于磷酸化介导的肌球蛋白运动活性调节很重要[Trybus, K. M., Freyzon, Y., Faust, L. Z., and Sweeney, H. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 74, 48 - 52]。为了研究S2处的特定氨基酸残基，或者杆状区域α-螺旋卷曲螺旋结构中出现的7残基/28残基重复序列的对齐对于这种相互作用是否至关重要，构建了两个平滑肌肌球蛋白突变体，其中S2的N端序列在不同程度上被删除。一个突变体在直接连接S1结构域与杆状下游序列的不变脯氨酸（Pro849）紧邻的C端位置缺失了71个残基，而在另一个突变体中，在Pro849下游56个残基的位置缺失了53个残基。尽管这些改变改变了肌球蛋白杆状序列中28残基重复序列和7残基重复序列的对齐，但通过电子显微镜观察，两个肌球蛋白突变体均显示出稳定的双头结构。突变体的肌动蛋白激活的ATP酶活性和肌动蛋白转运活性均完全受调节轻链磷酸化的调控。两个突变型肌球蛋白的肌动蛋白滑动速度与野生型重组肌球蛋白相同。此外，对于肌球蛋白丝形成至关重要的头部构象（伸展或折叠）在任何一个突变体中均未改变。这些结果表明，S2中的特定氨基酸残基或氨基酸重复序列的对齐对于头部构象均不重要。这些结果表明，头部-杆状连接处的特定氨基酸序列或杆状序列对齐对于平滑肌肌球蛋白的调节均不重要。
