Structure-Function Analysis of the C-terminal Domain of CNM67, a Core Component of the Saccharomyces cerevisiae Spindle Pole Body
Structure-Function Analysis of the C-terminal Domain of CNM67, a Core Component of the Saccharomyces cerevisiae Spindle Pole Body
复制标题
DOI：
10.1074/jbc.m111.227371
复制
发表时间：
2011-05-20
影响因子：
4.8
通讯作者：
Rayment, Ivan
中科院分区：
文献类型：
--
作者：
Klenchin, Vadim A.;Frye, Jeremiah J.;Rayment, Ivan
出芽酵母酿酒酵母（Saccharomyces cerevisiae）的纺锤体极体一直是用于理解微管组织中心的一个模型系统，但对其组成成分的分子结构知之甚少。我们在此报道核心成分Cnm67的C末端结构域在2.3埃分辨率下的结构。该结构的确定得益于一种针对含有卷曲螺旋的蛋白质结晶的新方法，这种方法利用球状结构域来稳定卷曲螺旋。这提高了它们在大肠杆菌中的溶解性并改善了其结晶情况。Cnm67的C末端结构域（残基Asn - 429 - Lys - 581）呈现出一种以前未曾见过的二聚体、交叉指状、全α - 螺旋折叠结构。体内研究表明，仅这个结构域就能够定位到纺锤体极体。此外，该结构揭示了一个在功能上不可或缺的大的带正电荷的表面区域，它与纺锤体极体的定位有关。最后，C末端的8个残基是无序的，但对蛋白质折叠和结构稳定性至关重要。
The spindle pole body of the budding yeast Saccharomyces cerevisiae has served as a model system for understanding microtubule organizing centers, yet very little is known about the molecular structure of its components. We report here the structure of the C-terminal domain of the core component Cnm67 at 2.3 angstrom resolution. The structure determination was aided by a novel approach to crystallization of proteins containing coiled-coils that utilizes globular domains to stabilize the coiled-coils. This enhances their solubility in Escherichia coli and improves their crystallization. The Cnm67 C-terminal domain (residues Asn-429-Lys-581) exhibits a previously unseen dimeric, interdigitated, all alpha-helical fold. In vivo studies demonstrate that this domain alone is able to localize to the spindle pole body. In addition, the structure reveals a large functionally indispensable positively charged surface patch that is implicated in spindle pole body localization. Finally, the C-terminal eight residues are disordered but are critical for protein folding and structural stability.