Local destabilization of the tropomyosin coiled coil gives the molecular flexibility required for actin binding.
Local destabilization of the tropomyosin coiled coil gives the molecular flexibility required for actin binding.
复制标题
原肌球蛋白卷曲线圈的局部不稳定提供了肌动蛋白结合所需的分子灵活性。
DOI：
10.1021/bi0348462
复制
发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Hitchcock-DeGregori,SarahE
中科院分区：
文献类型：
--
作者：
Singh,Abhishek;Hitchcock-DeGregori,SarahE
原肌球蛋白是一种沿着肌动蛋白丝长度结合的卷曲螺旋蛋白，含有40个不间断的卷曲螺旋特征的七肽重复序列。然而，它是灵活的。原肌球蛋白的区域，可能是重要的结合到细丝和肌钙蛋白相互作用偏离典型的卷曲螺旋结构在微妙的方式，改变局部构象或能量，而不中断卷曲螺旋。在富含界面丙氨酸（Ala簇）的区域中，链比在典型卷曲螺旋中更紧密地堆积，并且是交错的，导致弯曲[Brown等人（2001）Proc. Natl. Acad. Sci. U.S.A. 98，8496 - 8501]。Brown等人认为丙氨酸簇的弯曲使得原肌球蛋白缠绕在肌动蛋白丝螺旋上。另一种解释是卷曲螺旋的局部不稳定，而不是在Ala簇本身处的链的紧密堆积，允许柔性。将三个Ala残基改变为典型的界面残基A74 L − A78 V − A81 L，极大地稳定了原肌球蛋白，使用圆二色性和差示扫描量热法测量，并降低了肌动蛋白亲和力>10倍。正常的肌动蛋白亲和力和稳定性在突变体A74 Q-A78 N-A81 Q中恢复，该突变体模拟了Ala簇的稳定性，但没有模拟链的紧密堆积。接近N-末端引入的可比突变的分析和建模表明，对稳定性和功能的影响取决于背景。基于原肌球蛋白晶体结构的模型可以深入了解突变对结构的可能影响。我们的结论是，在允许原肌球蛋白的灵活性的丙氨酸簇的意义是稳定驱动的。
Tropomyosin, a coiled coil protein that binds along the length of actin filaments, contains 40 uninterrupted heptapeptide repeats characteristic of coiled coils. Yet, it is flexible. Regions of tropomyosin that may be important for binding to the filament and for interacting with troponin deviate from canonical coiled coil structure in subtle ways, altering the local conformation or energetics without interrupting the coiled coil. In a region rich in interface alanines (an Ala cluster), the chains pack closer than in canonical coiled coils, and are staggered, resulting in a bend [Brown et al. (2001)Proc. Natl. Acad. Sci. U.S.A. 98, 8496−8501]. Brown et al. suggested that bends at alanine clusters allow tropomyosin to wind on the actin filament helix. Another explanation is that local destabilization of the coiled coil, rather than close packing of the chains at Ala clusters per se, allows flexibility. Changing three Ala residues to canonical interface residues, A74L−A78V−A81L, greatly stabilized tropomyosin, measured using circular dichroism and differential scanning calorimetry, and reduced actin affinity >10-fold. Normal actin affinity and stability were restored in a mutant A74Q−A78N−A81Q that mimicked the stability of the Ala cluster but not the close packing of the chains. Analysis and modeling of comparable mutations introduced closer to the N-terminus show that the effects on stability and function depend on context. Models based on tropomyosin crystal structures give insight into possible effects of the mutations on the structure. We conclude that the significance of the Ala clusters in allowing flexibility of tropomyosin is stability-driven.