Abstract
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.
Highlights
ParF and ParG mediate TP228 plasmid segregation
ParF-ATP polymers provide a mechanism for plasmid segregation
Sedimentation Assays—Wild-type or mutant ParF proteins (6 – 8 M) in 30 mM Tris HCl, 100 mM KCl, 2 mM DTT, and 10% glycerol were incubated in the absence or presence of nucleotides (2 mM) and MgCl2 (5 mM) for 10 min at 30 °C
Summary
ParF and ParG mediate TP228 plasmid segregation. Results: ATP binding to ParF activates segregation, and ADP binding to ParF antagonizes its segregation function. Mutation of interface residues prevents both polymerization and DNA segregation in vivo 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. How ATP binding mediates partition by type Ib NTPases is currently unclear Data indicate that both P1 ParA and ␦ form polymers in an ATP-dependent manner. TP228 is a low copy number plasmid but exhibits no detectable loss during ϳ25 generations of unselected growth in E. coli [20] This segregation stability was shown to require a type Ib partition system encoding two proteins, the 22-kDa ParF protein and the 8.6-kDa ParG protein [20, 22]. To understand the structural basis for this process, we performed structural, biochemical, and cellular studies on the ParF protein
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