Abstract
ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.
Highlights
ParA ATPases are a ubiquitous family of proteins associated with directed movement within the bacterial cell of other proteins with or without additional attached macromolecules, such as DNA.[1,2] They are best known for their role in plasmid and chromosome partitioning, where they work with a ParB protein that binds to a centromerelike sequence within the DNA molecule to be partitioned.[3,4] They are associated with directing the segregation of molecular machines, such as chemotaxis protein factories.[5]
Both forms were detected in each species, the ratios differed significantly: the number of IncC1:IncC2 monomers per log-phase bacterium was 1627:145 for E. coli (10:1) and was 496:487 for P. putida (1:1)
Comparative studies on the naturally occurring RK2 IncC1 and IncC2 proteins reported here show that the N-terminal domain (NTD) of IncC1 can profoundly influence the activity of IncC2, the shorter protein, despite the latter apparently being functional for active partitioning in vivo.[26]
Summary
ParA ATPases are a ubiquitous family of proteins associated with directed movement within the bacterial cell of other proteins with or without additional attached macromolecules, such as DNA.[1,2] They are best known for their role in plasmid and chromosome partitioning, where they work with a ParB protein that binds to a centromerelike sequence within the DNA molecule to be partitioned.[3,4] They are associated with directing the segregation of molecular machines, such as chemotaxis protein factories.[5] Their ability to bind and hydrolyse ATP apparently provides the energy required to drive this movement.[6] For actin-like ParM ATPases, it is proposed that ParM ATP is added to the outer ends of a ParM filament and that paired ParR–parC complexes (the centromere binding protein and DNA, respectively) bind to this and favour ATP hydrolysis to ADP, which loosens the binding, allowing the plasmid–ParR complexes to separate and follow the growing tips of the ParM filament.[7,8,9,10,11] There is a body of evidence demonstrating that ParB proteins can pair their binding sites[12] and that ParA proteins form filaments[13,14,15,16] and show dynamic movement within the bacterial cell.[16] The recent ParM–ParR–parC models referred to above may apply to these systems, but this is not yet clear
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