The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins

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.

Fig. 1

BfpD forms hexamers upon nucleotide binding. (A) Gel filtration chromatograms for BfpD purification in the presence or absence of MgCl₂ and ATP as indicated. The accompanying silver-stained gels show SDS-PAGE analysis of peak fractions. (B) Electron micrograph of negatively-stained BfpD showing ring-shaped particles approximately 11.5 nm in diameter. Individual subunits comprising the ring are visible in some of the particles. (C-E) Electron micrographs of frozen-hydrated BfpD showing the 6-fold symmetry of the BfpD subunits. Scale bar, 10 nm.

Fig. 2

ATPase activity of purified hexameric BfpD. ATPase assays were carried out at 37°C in 40 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5% glycerol, 5 mM ATP and 10 mM ZnCl₂ unless otherwise indicated. The divalent cation (A), time (B), BfpD concentration (C) and ATP concentration (D) dependence of the ATPase activity are shown. ATPase activity was determined by quantifying the release of P_i and V_max and K_m values were calculated as described under Experimental Procedures. The data shown are derived from triplicate assays and are representative of three or more assays using different enzyme preparations.

Fig. 3

Yeast two-hybrid analysis of binary interactions between fragments of the N-terminus of BfpE and BfpD. Yeast strain AH109 was co-transformed with expression vectors encoding GAL4 DNA-binding (pGBKT7) and activation (pGADT7) domains fused in frame to fragments of the N-terminus of BfpE and full-length BfpD. For purposes of clarity, only the Bfp protein encoded on vectors pGBKT7 and pGADT7, respectively, is indicated. Numbers correspond to amino acid residue numbering of BfpE. + and – are the positive and negative controls, respectively, described in the text. Transformants were plated on medium containing X-alpha-Gal and lacking histidine and adenine and assayed for alpha-galactosidase activity to detect transcriptional activation of reporter genes MEL1, HIS3 and ADE2. Transformants that produced blue colonies on selective medium are shown in white, while those that did not grow are shown in black. Columns denote the mean ± SEM alpha-galactosidase activity from three experiments, each performed in triplicate.

Fig. 4

Titrations of hexameric BfpD into BfpE_39-76 (A) and BfpE_77-114 (B) in the presence of ATPγS (black) and ADP (red). Top panels show the raw data for the calorimetric titrations. The area under each peak represents the heat produced at each injection. The lower panels show integrated areas that were corrected for heat of dilution and plotted against the molar ratio (BfpD/peptide), with the best-fit curve for a one set of sites model.

Fig. 5

Binding of TNP derivatives of ATP and ADP to BfpD as monitored by enhancement of fluorescence. Monomeric BfpD was incubated with various concentrations of TNP-ATP (▵) and TNP-ADP (▴) and the fluorescence enhancement (ΔF) was measured (excitation 408 nm, emission 535 nm). Binding curves were fitted to an equation describing binding to a single affinity site (solid lines), and values for the dissociation constant and the maximum fluorescence enhancement (ΔF_max) were extracted. Results of three independent titrations are included.

Fig. 6

BfpD-ATPγS and BfpD-ADP binding to BfpE_77-114 and BfpE_39-76, respectively, enhances the proteinase K sensitivity of BfpD. Hexameric BfpD, alone and together with BfpE_39-76 or BfpE_77-114, in the presence of ATPγS (top panel) or ADP (bottom panel), was incubated with the indicated concentrations of proteinase K. Proteolytic digests were subjected to SDS-PAGE and visualized by silver staining.

Fig. 7

BfpC and BfpE increase the ATPase activity of hexameric BfpD by more than 1000-fold. BfpD ATPase assays were carried out in 40 mM Tris-HCl, pH 7.0, 150 mM NaCl, 5% glycerol, 5 mM ATP and 10 mM ZnCl₂, in the presence of the N-terminus of BfpC, BfpE_39-76 and BfpE_77-114 unless otherwise indicated (see Experimental Procedures). In (A), assays were performed in the presence of the indicated combination of BfpC, BfpE_39-76 and BfpE_77-114. In (B), assays were performed in the presence of BfpC and BfpE_77-114 and the Zn²⁺ in the reaction buffer was replaced with the indicated ion. The results are derived from at least three assays performed in triplicate, each using different BfpD preparations.

Fig. 8

Schematic diagram of the proposed mechanism by which BfpD transduces mechanical energy to the BFP biogenesis machine. Hexameric BfpD-ATP binds to the N-terminus of BfpC and to the last third of the N-terminus of BfpE. This greatly increases its ATPase activity and the resulting Bfp-ADP releases the distal third of the N-terminus of BfpE and instead binds to the middle third, while remaining bound to the N-terminus of BfpC. This movement drives the distal third of the BfpE N-terminus through the membrane. Upon replacement of ADP with ATP, BfpE relaxes, reestablishing its resting state.