Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites

The accurate segregation of chromosomal and extrachromosomal DNA requires specialized molecular machines, both in procaryotes and in eucaryotes (8, 19, 38, 41). For bacterial low-copy-number plasmids, the segrosome is the nucleoprotein complex that ensures their precise segregation to daughter cells at cytokinesis (13, 19, 20, 38, 39). Segrosomes can be categorized into subtypes based on their molecular components (39), but the best-characterized complexes consist of either a Walker (ParA) or actin-type ATPase, a cis-acting centromere site, and a centromere binding factor. ATP-mediated assembly of the actin-like ATPase into a bipolar spindle elicits bidirectional polymer growth, forcing the attached plasmids in opposite directions (15). Evidence is accumulating that the widespread ParA-type plasmid segregation ATPases also polymerize in response to nucleotide binding and that this polymerization mediates plasmid segregation. However, the molecular mechanisms that underpin this behavior and how plasmid segregation is achieved remain to be fully unraveled (1, 3, 5, 12, 14, 26, 27). The tripartite segrosome of multiresistance plasmid TP228 consists of the ParA homolog, ParF, and the ParG centromere binding protein, which assemble on the parH centromere (4, 18). ParF (22.0 kDa) is a member of the ParA superfamily of segregation proteins that are widely encoded by eubacterial and archaeal chromosomes and plasmids. Like its homologs, ParF is a weak ATPase whose nucleotide hydrolysis is enhanced by the partner protein, ParG (5). Strikingly, ATP binding promotes the polymerization of ParF into extensive multistranded filaments. ParG (8.6 kDa) enhances ParF polymerization independently of ATP but also superstimulates filamentation in the presence of ATP. In contrast, ParF polymerization is blocked by ADP (3, 5). This suggests that ParF action during partitioning may involve a cycle of polymerization and depolymerization in which, following segrosome formation at the centromere, the binding of ATP and ParG initially augments ParF filamentation. Stimulation of ParF nucleotide hydrolysis by ParG subsequently may induce the formation of ParF-ADP species within polymers, blocking further filament growth. Pushing of plasmids by ParF polymer extension or plasmid pulling by filament depolymerization may drive replicated plasmids to either side of the septal plane (5). ParG is a homodimeric DNA binding protein (4) with C-terminal regions that interweave into a ribbon-helix-helix (RHH) fold and mobile N-terminal tails (16). Dimerization, DNA binding at the operator site, and interaction with ParF are mediated by the C-terminal regions (3, 7, 16). The flexible N termini are also multifunctional. First, the N-terminal tail of ParG includes an arginine finger-like motif that enhances ATP hydrolysis by ParF (3). The motif may be part of a semiflexible loop that intercalates into the ParF nucleotide binding pocket, analogous to arginine fingers in proteins such as human Ras-GAPs (2, 31). Arginine finger loops stabilize the transition state during nucleotide hydrolysis by their partner proteins (6), and the same may be the case with ParF-ParG. Stimulation of nucleotide hydrolysis by ParG may be an important step in the ParF polymerization-depolymerization cycle (3). Second, and separately from its role in ATPase enhancement, the ParG mobile tail is required for stimulation of ParF polymerization (3). ParG may either bundle filaments more extensively or stabilize ParF protomers within filaments (3, 5). The flexible tails within each ParG dimer potentially enwrap ParF monomers, either on the same or on adjacent protofilaments, or might act at points of polymer disassembly. Thus, ParG may be functionally analogous to microtubule-associated proteins that regulate tubulin kinetics or to formins and other factors that modulate growth and retraction of eucaryotic actin filaments (27). Third, ParG is a transcriptional repressor of the parFG genes (7), with antiparallel β-strands thought to insert into the major groove at the operator site as established for other RHH proteins (17, 34, 36, 42). The tail modulates the interaction of ParG with the operator site (here designated OF) during autoregulation. Specifically, a transient β-strand element in the tail that associates with the RHH domain is implicated in formation of the repressive nucleoprotein complex (7). Here, we probe further the interaction between ParG and the operator: the locus comprises a complex set of related motifs that ParG recognizes with different affinities, suggesting that the operator has evolved with sites that bind ParG dissimilarly to generate a nucleoprotein complex that is optimized for parFG regulation.