The ATPase Activity of BfpD Is Greatly Enhanced by Zinc and Allosteric Interactions with Other Bfp Proteins.

Type IV pilus biogenesis, protein secretion, DNA transfer, and filamentous phage morphogenesis systems are thought to possess similar architectures and mechanisms. These multiprotein complexes include members of the Pule superfamily of putative NTPases that have extensive sequence similarity and probably similar functions as the energizers of macromolecular transport. We purified the Pule homologue BfpD of the enteropathogenic Escherichia coli bundle-forming pilus (BFP) biogenesis machine and characterized its ATPase activity, providing new insights into its mode of action. Numerous techniques revealed that BfpD forms hexamers in the presence of nucleotide. Hexameric BfpD displayed weak ATPase activity. We previously demonstrated that the N termini of membrane proteins BfpC and BfpE recruit BfpD to the cytoplasmic membrane. Here, we identified two BfpD-binding sites, BfpE39-76 and BfpE77-l14, in the N terminus of BfpE using a yeast two-hybrid system. Isothermal titration calorimetry and protease sensitivity assays showed that hexameric BfpD-ATPγS binds to BfoE77-114, whereas hexameric BfpD-ADP binds to BfpE39-76. Interestingly, the N terminus of BfpC and BfoE77-114 together increased the ATPase activity of hexameric BfpD over 1200-fold to a Vmax of 75.3 µmol of Pi min-1 mg-1, which exceeds by over 1200-fold the activity of other Pule family members. This augmented activity occurred only in the presence of Zn2+. We conclude that allosteric interactions between BfpD and BfpC and BfpE dramatically stimulate its ATPase activity. The differential nucleotide-dependent binding of hexameric BfpD to BfpE39-76 and BfpE77-114 suggests a model for the mechanism by which BfpD transduces mechanical energy to the biogenesis machine. [ABSTRACT FROM AUTHOR]