The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram‐negative bacteria. We reported previously the structure of the ADP‐bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N‐terminal domains exhibit a collection of rigid‐body conformations. Nucleotide binding ‘locks’ the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide‐dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Bio chemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.

The DNA‐binding domain of the Escherichia coli DnaA protein is represented by the 94 C‐terminal amino acids (domain 4, aa 374–467). The isolated DNA‐binding domain acts as a functional repressor in vivo, as monitored with a mioC::lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site‐directed and random PCR mutagenesis were performed, using the mioC::lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA‐binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N‐terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N‐terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non‐specifically.

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