TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase

Abstract

Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwB⌬N70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwB⌬N70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwB⌬N70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwB⌬N70 -DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F 1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor. molecular motor ͉ DNA transfer C onjugative plasmids provide a main route for acquisition of new genetic information in bacteria. Bacterial conjugation systems (including the related Vir system that Agrobacterium tumefaciens uses to transfer DNA to plants) show similarities to both DNA replication and protein transport systems (1, 2). The translocated substrate is a nucleoprotein particle that crosses the bacterial envelope into other bacterial or eukaryotic cells, crossing the kingdom boundaries. A two-step mechanism for DNA transport was proposed (2), in which conjugation is visualized as a DNA rolling-circle replication process linked to a type IV protein secretion system. Conjugation is encoded by two gene clusters in most conjugative plasmids. One cluster (dtr) encodes the DNA transfer replication proteins, and the other (mpf ) codes for the proteins that assemble the type IV protein secretion system channel. R388 is a 34-kb plasmid, which displays the simplest known dtr region, encoding only three proteins (TrwA, TrwB, and TrwC). The mpf region codes for 11 proteins (TrwD-N) involved in the formation of an extracellular pilus and a membrane-associated complex that translocates the nucleoprotein substrate across membranes.TrwB, an integral membrane and DNA-binding protein, is a key player in R388 conjugation (3-6). It has a counterpart in all conjugative systems, and it is thought to be responsible for recruiting the relaxosome DNA-pr...

Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

“…identified as a DNA-dependent ATPase (Tato et al, 2005). The discovery that a coupling protein is able to hydrolyse ATP substantiates the finding that key, well-conserved residues in the Walker A and B regions are necessary for conjugative transfer.…”

“…The SpoIIIE protein has DNA-dependent ATP hydrolysis activity that is used to pump DNA during sporulation (Bath et al, 2000). The R388 coupling protein also has DNA-dependent ATP hydrolysis activity, and the Walker motifs of R388, RP4 and F plasmid have been demonstrated to be involved in conjugation (Moncalian et al, 1999;Schroder et al, 2002;Tato et al, 2005). Mutational analyses of the Walker A and B motifs of the RP4 coupling protein, TraG, revealed that only two mutations completely abolished RP4 transfer: K187T in Walker A, and D449N in Walker B (Balzer et al, 1994).…”

“…In addition, coupling proteins have conserved motifs such as the Walker-type nucleoside triphosphate (NTP)-binding domains. Recent analysis of TrwB has revealed that this coupling protein is a DNA-dependent ATPase (Tato et al, 2005). Mutational analyses of the Walker motifs of TraG, the coupling protein from RP4, indicated that the Lys and Asp residues, at positions 187 and 449 of RP4, were essential for conjugation of the IncP plasmid (Balzer et al, 1994).…”

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli

“…Extrusion thus is unlikely to occur unless the neighboring protomer binds the second ligand, and we are led to anticipate a positive cooperative kinetics in transport. Positive cooperativity was indeed reported for ATP hydrolysis in the dimeric MalK in the maltose transporter complex (24) and in the hexameric ATPase TrwB involved in DNA transfer in conjugation (25), although kinetic analysis has been difficult with the classical F 1 ATPase (26,27). Although it was disappointing to find no evidence for positive cooperativity for nitrocefin, there was ample evidence for that for cephalothin, cefamandole, and cephaloridine.…”

Autoinhibitory Regulation of TrwK, an Essential VirB4 ATPase in Type IV Secretion Systems

“…In both cases, the template was TrwB, an hexameric integral membrane protein encoded by the conjugative plasmid R388 that is involved in the movement of the relaxosome DNA-binding complex toward the T4SS (28). TrwB belongs to the RecA protein-like family (26) and it displays a DNA-dependent ATPase activity (10,29,30). Likewise, TrwK, also encoded by plasmid R388, is also able to hydrolyze ATP (8).…”