Department of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center, Kansas City, KS 66160.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160.
Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):7497-7504. doi: 10.1073/pnas.1707385114. Epub 2017 Jun 26.
In MinE induces MinC/MinD to oscillate between the ends of the cell, contributing to the precise placement of the Z ring at midcell. To do this, MinE undergoes a remarkable conformational change from a latent 6β-stranded form that diffuses in the cytoplasm to an active 4β-stranded form bound to the membrane and MinD. How this conformational switch occurs is not known. Here, using hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) we rule out a model in which the two forms are in rapid equilibrium. Furthermore, HDX-MS revealed that a MinE mutant (D45A/V49A), previously shown to produce an aberrant oscillation and unable to assemble a MinE ring, is more rigid than WT MinE. This mutant has a defect in interaction with MinD, suggesting it has difficulty in switching to the active form. Analysis of intragenic suppressors of this mutant suggests it has difficulty in releasing the N-terminal membrane targeting sequences (MTS). These results indicate that the dynamic association of the MTS with the β-sheet is fine-tuned to balance MinE's need to sense MinD on the membrane with its need to diffuse in the cytoplasm, both of which are necessary for the oscillation. The results lead to models for MinE activation and MinE ring formation.
在 MinE 的作用下,MinC/MinD 在细胞两端之间振荡,有助于将 Z 环精确地定位于细胞中部。为了实现这一点,MinE 经历了一个显著的构象变化,从在细胞质中扩散的潜伏的 6β 股形式转变为与膜和 MinD 结合的活性 4β 股形式。这种构象开关是如何发生的尚不清楚。在这里,我们使用氢氘交换结合质谱(HDX-MS)排除了两种形式处于快速平衡的模型。此外,HDX-MS 显示,先前显示产生异常振荡且无法组装 MinE 环的 MinE 突变体(D45A/V49A)比 WT MinE 更具刚性。该突变体与 MinD 的相互作用有缺陷,表明其难以转换为活性形式。对该突变体的基因内抑制因子的分析表明,它难以释放 N 端膜靶向序列(MTS)。这些结果表明,MTS 与 β 片层的动态结合被精细地调节,以平衡 MinE 在膜上感应 MinD 的需求与其在细胞质中扩散的需求,这两者都是振荡所必需的。这些结果为 MinE 的激活和 MinE 环的形成提供了模型。