Abstract
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, BfpE(39-76) and BfpE(77-114), in the N terminus of BfpE using a yeast two-hybrid system. Isothermal titration calorimetry and protease sensitivity assays showed that hexameric BfpD-ATPgammaS binds to BfpE(77-114), whereas hexameric BfpD-ADP binds to BfpE(39-76). Interestingly, the N terminus of BfpC and BfpE(77-114) together increased the ATPase activity of hexameric BfpD over 1200-fold to a V(max) of 75.3 mumol of P(i) 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 Zn(2+). 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 BfpE(39-76) and BfpE(77-114) suggests a model for the mechanism by which BfpD transduces mechanical energy to the biogenesis machine.
Highlights
In this paper we describe a detailed study of the ATPase activity of BfpD in which we address its activity in the context of the bundle-forming pilus (BFP) biogenesis machine, i.e. in the presence of components of the machinery with which it interacts
BfpD Is a Hexamer—BfpD synthesized with a N-terminal His tag in E. coli strain BL21pLysS,pRPA405 was purified to homogeneity using two chromatographic steps
BL21pLysS,pRPA405 produces functional BfpD as plasmid pRPA405, encoding the His-tagged BfpD, complements the bfpD mutant UMD926 to restore autoaggregation, a phenotype that correlates with biogenesis of functional BFP
Summary
Type IV pili; BFP, bundle-forming pilus; DLS, dynamic light scattering; EPEC, enteropathogenic Escherichia coli; ⌬H, enthalpy change; ITC, isothermal titration calorimetry; Kav, partition coefficient; s20,w, sedimentation coefficient; SV40, simian virus 40; TNP, 2Ј,3Ј-O-trinitrophenyl; X-Gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; Mops, 4-morpholinepropanesulfonic acid; ATP␥S, adenosine 5Ј-O-(3-thiotriphosphate). BfpD is a member of the PulE superfamily of putative nucleotide-binding proteins involved in bacterial type II and IV secretion, Tfp assembly, natural competence, and assembly of archeal flagellae [17]. Mutation of key residues in the Walker A box of PulE superfamily members abolishes activity of the cognate systems, for example, PulE [39, 40], EpsE [41], OutE [42], PilB [43], and BfpD [4] These proteins most likely share the function of supplying energy from ATP hydrolysis to drive organellar assembly or substrate translocation. The results of our studies suggest a model for conversion of chemical energy into mechanical energy to power extraction of bundlin from the cytoplasmic membrane for assembly into BFP filaments
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