The intermediate filament protein consensus motif of helix 2B:: Its atomic structure and contribution to assembly
The intermediate filament protein consensus motif of helix 2B:: Its atomic structure and contribution to assembly
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DOI：
10.1006/jmbi.2000.3719
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发表时间：
2000-05-19
影响因子：
5.6
通讯作者：
Aebi, U
中科院分区：
文献类型：
--
作者：
Herrmann, H;Strelkov, SV;Aebi, U
几乎所有的中间丝蛋白在它们的中央α-螺旋杆结构域的C-末端都表现出高度保守的氨基酸基序（YRKLLEGEE）。我们已经分析了其贡献的各个阶段的装配，通过使用截短形式的非洲爪蟾波形蛋白和小鼠结蛋白，VimIAT和DesIAT，终止之前，这个主题，通过比较它们与野生型和无尾蛋白。令人惊讶的是，在全长蛋白质形成四聚体的低离子强度和高pH的缓冲液中，VimIAT和DesIAT结合成各种高分子量复合物。组装开始后，VimIAT和DesIAT聚集成单位长度型细丝，其快速纵向退火以产生直径约20 nm的细丝。通过扫描透射电子显微镜的质量测量结果表明，VimIAT和DesIAT长丝包含相当多的亚基每个横截面比标准的中间丝。这表明YRKLLEGEE基序对于真正的四聚体复合物的形成以及组装过程中控制细丝宽度而不是伸长至关重要。为了确定YRKLLEGEE结构域的结构，我们生长了含有螺旋2B的最后28个氨基酸残基的肽晶体，在其氨基末端嵌合融合到酵母转录因子GCN 4的31个氨基酸长的亮氨酸拉链结构域，以促进适当的卷曲螺旋形成。原子结构表明，从Tyr 400开始，两个螺旋逐渐分离，卷曲螺旋终止于残基Glu 405，而下游残基折叠远离卷曲螺旋轴。(C)北京大学出版社.
Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central alpha-helical rod domain. We have analyzed its contribution to the various stages of assembly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them with the wild-type and tailless proteins. It is surprising that in buffers of low ionic strength and high pH where the full-length proteins form tetramers, both VimIAT and DesIAT associated into various high molecular weight complexes. After initiation of assembly, both VimIAT and DesIAT aggregated into unit-length-type filaments, which rapidly longitudinally annealed to yield filaments of around 20 nm in diameter. Mass measurements by scanning transmission electron microscopy revealed that both VimIAT and DesIAT filaments contained considerably more subunits per cross-section than standard intermediate filaments. This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control of filament width, rather than elongation, during assembly. To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amino-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation. The atomic structure shows that starting from Tyr400 the two helices gradually separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis. (C) 2000 Academic Press.