Vimentin Coil 1A-A Molecular Switch Involved in the Initiation of Filament Elongation
Vimentin Coil 1A-A Molecular Switch Involved in the Initiation of Filament Elongation
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DOI：
10.1016/j.jmb.2009.04.067
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发表时间：
2009-07-10
影响因子：
5.6
通讯作者：
Burkhard, Peter
中科院分区：
文献类型：
--
作者：
Meier, Markus;Padilla, G. Pauline;Burkhard, Peter
有趣的是，我们之前发表的人类中间丝蛋白波形蛋白的卷曲1A片段的结构被证明是一个单体α螺旋卷曲，而不是预期的二聚螺旋卷曲。然而，39个氨基酸长的螺旋具有与卷曲线圈相容的固有曲率。我们现在已经设计了波形蛋白线圈1A的四个突变体，修改了七肽重复模式中的关键a和d位置，目的是研究稳定二聚体螺旋线圈结构所需的分子标准。用圆二色谱、分析超速离心法和X射线结晶学对突变体的生物物理性质进行了分析。所有四个突变体都表现出比野生型更稳定的表现，这表明熔化温度(T-m)上升。在0.1 mg/ml浓度下，单点突变Y117L多肽的T-m比野生型提高了46℃。一般来说，在a或d位置引入一个稳定点突变确实会诱导稳定的二聚体的形成，这一点通过沉降平衡实验得到了证明。X射线结晶学进一步证实了Y117L多肽的二聚体齐聚状态，得到了真正的盘绕几何结构。最值得注意的是，当这种突变被引入全长波形蛋白时，细丝组装在单位长度细丝(ULF)水平上完全停止，无论是在体外还是在转基因的培养细胞中都是如此。因此，野生型线圈1A形成稳定的双股线圈的低倾向很可能是将超低密度纤维端到端退火成细丝的先决条件。相应地，线圈1A结构域可能从二聚体α-螺旋线圈“切换”到更开放的结构，从而在ULF中调节中间细丝延长反应所需的四聚体亚基的构象重排。(C)2009爱思唯尔有限公司。保留所有权利。
Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric alpha-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T-m). At a concentration of 0.1 mg/ml, the T-m of the peptide with the single point mutation Y117L increased dramatically by 46 degrees C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by Xray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might "switch" from a dimeric alpha-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the intermediate filament elongation reaction. (C) 2009 Elsevier Ltd. All rights reserved.