School of Life and Environmental Sciences, University of Sydney, New South Wales 2006, Australia.
Microbiology and Infectious Diseases, School of Medicine, Western Sydney University, New South Wales 2751, Australia; Antibiotic Resistance & Mobile Elements Group, Ingham Institute for Applied Medical Research, New South Wales 2170, Australia.
J Mol Biol. 2022 Oct 15;434(19):167770. doi: 10.1016/j.jmb.2022.167770. Epub 2022 Jul 27.
The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.
原核质体的分离通常需要一个类似于着丝粒的位点和两种蛋白质，即着丝粒结合蛋白（CBP）和 NTP 酶。相比之下，单个 245 个残基的 Par 蛋白在没有可识别的 NTP 酶成分的情况下，介导了典型的葡萄球菌多耐药质体 pSK1 的分配。为了深入了解 pSK1 Par 对着丝粒的结合及其分离功能，我们进行了结构、生化和体内研究。在这里，我们显示 pSK1 Par 结合由七个重复元件组成的着丝粒。我们证明这种 Par-着丝粒相互作用也介导 Par 自身调节。为了阐明 Par 着丝粒结合机制，我们获得了 Par N 端 DNA 结合结构域与 2.25Å 的着丝粒 DNA 结合的结构。pSK1 Par 结构含有一个翼状螺旋-转角-螺旋（wHTH），与其他质体 CBP 结构不同，但与 B. subtilis 染色体分离蛋白 RacA 具有同源性。生化研究表明，Par wHTH 结构域 C 端形成卷曲螺旋，并介导寡聚化。荧光显微镜分析表明，pSK1 Par 增强了质粒与簇的分离，在细胞分裂时促进有效分离。综合这些数据，深入了解了单个蛋白分配系统的分子特性。
