Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition

Abstract

The segregation of plasmid DNA typically requires three elements: a DNA centromere site, an NTPase, and a centromere-binding protein. Because of their simplicity, plasmid partition systems represent tractable models to study the molecular basis of DNA segregation. Unlike eukaryotes, which utilize the GTPase tubulin to segregate DNA, the most common plasmid-encoded NTPases contain Walker-box and actin-like folds. Recently, a plasmid stability cassette on Bacillus thuringiensis pBtoxis encoding a putative FtsZ/tubulin-like NTPase called TubZ and DNA-binding protein called TubR has been described. How these proteins collaborate to impart plasmid stability, however, is unknown. Here we show that the TubR structure consists of an intertwined dimer with a winged helix-turn-helix (HTH) motif. Strikingly, however, the TubR recognition helices mediate dimerization, making canonical HTH-DNA interactions impossible. Mutagenesis data indicate that a basic patch, encompassing the two wing regions and the N termini of the recognition helices, mediates DNA binding, which indicates an unusual HTH-DNA interaction mode in which the N termini of the recognition helices insert into a single DNA groove and the wings into adjacent DNA grooves. The TubZ structure shows that it is as similar structurally to eukaryotic tubulin as it is to bacterial FtsZ. TubZ forms polymers with guanine nucleotide-binding characteristics and polymer dynamics similar to tubulin. Finally, we show that the exposed TubZ C-terminal region interacts with TubR-DNA, linking the TubR-bound pBtoxis to TubZ polymerization. The combined data suggest a mechanism for TubZ-polymer powered plasmid movement.

One-way ticket to the cell pole: Plasmid transport by the prokaryotic tubulin homolog TubZ

“…The authors show that TubR forms a highly intertwined dimer. Each monomer harbors five α-helices and three β-ribbons arranged in the sequence 5 , which contains a winged helix-turn-helix (HTH) motif structurally similar to that found in bacterial transcriptional repressors of the ArsR family (10). This finding is very intriguing, as to date no DNA segregation protein has been shown to be homologous to the metal-binding transcription factors of the ArsR group.…”

“…The overlay highlights similarities in the winged HTH motif. However, four key differences emerge: (i) TubR forms a dimer that is remarkably distinct from that formed by CzrA; (ii) TubR does not contain the metal-binding motif typical of either the ArsR proteins or any other metal-regulated factor; (iii) the monomermonomer interface is extremely different in the TubR dimer compared with the CzrA dimer; and (iv) the recognition helices of the HTH domain are solvent exposed in CzrA, whereas they are largely buried in the TubR dimer (10). This last observation raises a unique theme: the recognition helices of the HTH motif mediate dimerization of TubR and in doing so, they remain buried in the monomermonomer interface, leaving only some N-terminal residues accessible for other interactions.…”

“…The report by Ni et al describes the structure of the TubR protein, the centromere-binding component of the TubRZ system (10). The authors show that TubR forms a highly intertwined dimer.…”

“…Type III systems were identified more recently: this discovery has opened up unforeseen perspectives on prokaryotic cytoskeletal proteins mediating genome segregation (7)(8)(9). In PNAS, Ni et al shed light on the molecular mechanisms underpinning the function of type III partition cassettes by reporting the structures of TubR and TubZ proteins encoded by the pBtoxis plasmid from Bacillus thuringiensis (10).…”

“…The Schumacher team has identified a large basic patch on the surface of TubR: this region includes residues Arg-74, Arg-77, and Lys-79 located in the wing (the loop between β 2 and β 3 ) adjacent to the HTH and Lys-43 in the α 3 helix preceding the recognition helix, α 4 , in the HTH signature (10). A mutational analysis confirmed that these positively charged residues are crucial for DNA binding, indicating that this patch represents the bona fide DNA-binding domain of TubR.…”