Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, USA.
J Biol Chem. 2012 Jul 27;287(31):26146-54. doi: 10.1074/jbc.M112.373696. Epub 2012 Jun 6.
Segregation of the bacterial multidrug resistance plasmid TP228 requires the centromere-binding protein ParG, the parH centromere, and the Walker box ATPase ParF. The cycling of ParF between ADP- and ATP-bound states drives TP228 partition; ATP binding stimulates ParF polymerization, which is essential for segregation, whereas ADP binding antagonizes polymerization and inhibits DNA partition. The molecular mechanism involved in this adenine nucleotide switch is unclear. Moreover, it is unknown how any Walker box protein polymerizes in an ATP-dependent manner. Here, we describe multiple ParF structures in ADP- and phosphomethylphosphonic acid adenylate ester (AMPPCP)-bound states. ParF-ADP is monomeric but dimerizes when complexed with AMPPCP. Strikingly, in ParF-AMPPCP structures, the dimers interact to create dimer-of-dimer "units" that generate a specific linear filament. Mutation of interface residues prevents both polymerization and DNA segregation in vivo. Thus, these data provide insight into a unique mechanism by which a Walker box protein forms polymers that involves the generation of ATP-induced dimer-of-dimer building blocks.
细菌多药耐药质粒 TP228 的分离需要着丝粒结合蛋白 ParG、parH 着丝粒和 Walker 盒 ATP 酶 ParF。ParF 在 ADP 和 ATP 结合状态之间循环驱动 TP228 分区；ATP 结合刺激 ParF 聚合，这对于分离是必不可少的，而 ADP 结合拮抗聚合并抑制 DNA 分区。涉及此腺嘌呤核苷酸开关的分子机制尚不清楚。此外，尚不清楚任何 Walker 盒蛋白如何以 ATP 依赖性方式聚合。在这里，我们描述了 ADP 和磷酸甲基膦酸腺苷酸酯 (AMPPCP) 结合状态下的多种 ParF 结构。ParF-ADP 是单体，但与 AMPPCP 复合时会二聚化。引人注目的是，在 ParF-AMPPCP 结构中，二聚体相互作用以产生特定的线性细丝。突变界面残基可阻止体内聚合和 DNA 分离。因此，这些数据提供了对 Walker 盒蛋白形成聚合物的独特机制的深入了解，该机制涉及生成 ATP 诱导的二聚体构建块。
