The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif

Abstract

ParA superfamily ͉ polymerization ͉ plasmid partition ͉ ATPase T he precise distribution of newly replicated genomes to progeny cells is imperative for stable transmission of genetic information. In bacteria, the most well characterized segregation mechanisms are specified by low-copy-number plasmids. These systems most frequently comprise two plasmid-encoded proteins, often termed ParA and ParB, that assemble on a cis-acting centromeric site. ParB directly binds the centromere, whereas ParA is recruited by interactions with ParB. The resulting segrosome complex is a positioning apparatus that localizes the attached plasmids to specific subcellular addresses (1, 2).The segregation locus of multidrug-resistance plasmid TP228 in Escherichia coli consists of the parF and parG genes and nearby parH centromere (3). ParG (8.6 kDa) is the prototype of a class of small proteins involved in accurate segregation that are unrelated phylogenetically to ParB, but that fulfil analogous functions as centromere-binding factors (1,4,5). ParG is dimeric, with symmetric C-terminal domains that interleave into a ribbon-helixhelix fold that is crucial for DNA binding, and unstructured N-terminal tails (4, 6). Additional to its role as a centromerebinding protein, ParG is a transcriptional repressor of the parFG genes: transient associations between the flexible and folded domains in complex with target DNA modulate organization of a higher-order complex critical for transcriptional repression (7).The ParA superfamily of ATPases, widely encoded by both chromosomes and plasmids, is characterized by a variant Walkertype ATP-binding motif (8). ParF (22.0 kDa) epitomizes one clade of the superfamily (3). In common with other ParA proteins, ParF is a weak ATPase whose nucleotide hydrolysis is enhanced Ϸ30-fold by ParG (9). ATP binding and/or hydrolysis by ParA proteins has long been recognized as a crucial facet of the segregation process, although its mechanistic purpose was uncertain (10-12). We have recently shown that ATP binding stimulates the polymerization of ParF into extensive multistranded filaments, whereas ADP antagonizes filamentation. ParG is another key modulator of polymerization (9). Mutagenesis of the ATP-binding site in Parf perturbed DNA segregation in vivo, ATP hydrolysis, and polymerization. We envisage that segrosome formation is initiated by site-specific binding of ParG to parH, generating paired complexes of specific topology. ParF is then recruited. ParF polymerization within the complex is controlled by nucleotide binding, by ParG-mediated stimulation of ATP hydrolysis, by remodeling effects of ParG, and, more speculatively, by cell cycle signals. Polymerization, or depolymerization, invokes separation of paired plasmids and their segregation in opposite poleward directions (1, 9).Arginine fingers stimulate nucleotide hydrolysis by NTPases through the action of an arginine side chain inserted into the catalytic niche (13,14). The arginine stabilizes the transition state through neutralization of negative charge...

ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor

“…6 A and B). ParC has a weak intrinsic ATPase activity of V max = 4.3 mM ATP hydrolyzed·min −1 ·mol −1 protein and a K m = 20 μM ATP, which is similar to that of other Walker-type ATPases (15,26). Based on previous characterization of ParA mutants, we also engineered point mutations in ParC that are expected (i) to abolish ATP binding and lock ParC in the Apo-monomer state (ParCK15Q); or (ii) to enable ATP binding but inhibit hydrolysis and allow ParC to cycle between existence as an ATP-bound monomer and a 2ATP-bound dimer (ParCG11V).…”

“…Processes regulated by ParA-like proteins often are regulated by or are dependent on associated proteins that modulate ParA ATPase activity (17,18,26,(28)(29)(30)(31). It is possible that ParP similarly modulates ParC's enzymatic activity; to date, we have been unable to purify ParP to test this possibility.…”

Superstructure of the centromeric complex of TubZR C plasmid partitioning systems

“…Type I plasmid partitioning systems (4,5) are based on deviant Walker A ATPases (6,7), type II (8) on actin-like proteins (9), and type III (10)(11)(12) on tubulin/FtsZ-like proteins (13,14). Although the structure of the filament implicated in segregation has been determined for all three systems (9,(14)(15)(16), only examples of type I and II centromeric complexes have so far been resolved (17)(18)(19).…”

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes

“…ParB binds to the plasmid cargo to be segregated by binding to a specific site (parS) on the plasmid. When ParB interacts with ParA, it stimulates the ATPase activity of ParA, resulting in depolymerization of the ParA filament with basic residues in the N-terminal region of ParB responsible for the stimulation of ParA ATPase activity (6,10,11). In some systems, polymerization and depolymerization result in the ParA oscillating over the chromosome (8).…”

“…A related plasmid segregation system uses a ParA-like protein, ParF, which interacts with a protein unrelated to ParB, ParG. This, centromere-binding protein stimulates the ATPase activity of ParF via its N terminus (11). Proteins in other species show no sequence similarity to ParG but are able to act as functional equivalents (12).…”