FliG subunit arrangement in the flagellar rotor probed by targeted cross-linking
FliG subunit arrangement in the flagellar rotor probed by targeted cross-linking
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DOI：
10.1128/jb.187.16.5640-5647.2005
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发表时间：
2005-08-01
影响因子：
3.2
通讯作者：
Blair, DF
中科院分区：
文献类型：
--
作者：
Lowder, BJ;Duyvesteyn, MD;Blair, DF
FliG是细菌鞭毛转子上开关复合物的组分。每个鞭毛马达含有约25个FliG分子。大肠杆菌的蛋白质具有331个氨基酸残基并且包含至少两个离散结构域。约100个残基的C-末端结构域在旋转中起作用，并且包括与定子蛋白MotA相互作用的带电残基。FliG蛋白的其他部分对于鞭毛组装是必需的，并且与MS环蛋白FliF和开关复合物蛋白FliM相互作用。FliG的中间和C-末端部分的晶体结构显示由α-螺旋连接的两个球状结构域和含有两个高度保守的甘氨酸残基的短延伸片段。在这里，我们描述了有针对性的交联研究FliG，揭示其组织的鞭毛的功能。半胱氨酸残基被引入在不同的位置，单独或成对的，并通过马来酰亚胺或二硫化物诱导氧化剂的交联进行了检查。在中间结构域的某些位置具有成对Cys残基的FliG分子以高产率形成二硫键连接的二聚体和较大的多聚体，表明相邻亚基的中间结构域相当接近，并对结构域的相对取向施加限制。在C-末端结构域中具有单个Cys替换的某些蛋白质形成具有中等产量的二聚体，但不形成较大的多聚体。交联的结果和突变和电子显微镜研究的数据的基础上，我们提出了一个模型的鞭毛中的FliG亚基的组织。
FliG is a component of the switch complex on the rotor of the bacterial flagellum. Each flagellar motor contains about 25 FliG molecules. The protein of Escherichia coli has 331 amino acid residues and comprises at least two discrete domains. A C-terminal domain of about 100 residues functions in rotation and includes charged residues that interact with the stator protein MotA. Other parts of the FliG protein are essential for flagellar assembly and interact with the MS ring protein FliF and the switch complex protein FliM. The crystal structure of the middle and C-terminal parts of FliG shows two globular domains joined by an alpha-helix and a short extended segment that contains two well-conserved glycine residues. Here, we describe targeted cross-linking studies of FliG that reveal features of its organization in the flagellum. Cys residues were introduced at various positions, singly or in pairs, and cross-linking by a maleimide or disulfide-inducing oxidant was examined. FliG molecules with pairs of Cys residues at certain positions in the middle domain formed disulfide-linked dimers and larger multimers with a high yield, showing that the middle domains of adjacent subunits are in fairly close proximity and putting constraints on the relative orientation of the domains. Certain proteins with single Cys replacements in the C-terminal domain formed dimers with moderate yields but not larger multimers. On the basis of the cross-linking results and the data available from mutational and electron microscopic studies, we propose a model for the organization of FliG subunits in the flagellum.