Abstract
Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS.
Highlights
T4SSs are macromolecular assemblies composed of 11 mating pair proteins (VirB1 to VirB11) and a coupling protein (VirD4) that span inner and outer bacterial membranes
We demonstrated that TrwK, the VirB4 homologue in the R388 conjugative system, is able to hydrolyze ATP in the absence of potential substrates (8)
A template consisting of the atomic structure of TrwB coupling protein (1e9r.pdb) (26) is very similar to a previously reported model of a TrwK homologue, the VirB4 protein of A. tumefaciens (17)
Summary
Cloning of TrwK and Mutants—The DNA of R388 gene trwK was amplified by PCR and cloned into a pET3a expression vector (Novagen, Madison, WI). The trwK_1–772, trwK_1–787, and trwK_1– 801 mutants were generated by PCR using the same forward primer
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