Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli

Abstract

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 microM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.

Figure 1. Overproduction and purification of the His-tagged ABC ATPase domain of HlyB at 25 °C

(A) SDS/PAGE of total cell protein stained with Coomassie Brilliant Blue is shown. E. coli strain DH5α was transformed with plasmid pPSG122 expressing the ABC (His₆) domain. Lane 1, molecular-mass standards (in kDa, as indicated to the left of the gel); lane 2, total bacterial proteins before arabinose induction (loaded sample equivalent to an A₆₀₀ of 0.25); lane 3, after induction (loaded sample equivalent to an A₆₀₀ of 0.25); lane 4, cytoplasmic (soluble) fraction; lane 5, membrane plus insoluble aggregated fraction. Lanes 4 and 5 contain equivalent cell loadings. The arrow indicates the ABC ATPase domain of HlyB (28 kDa). (B) Peak fractions eluted from the Sepharose–Ni²⁺, separated and stained with Coomassie Brilliant Blue. Lane 1, molecular-mass standards (in kDa, as indicated to the left of the gel); lanes 2–5, imidazole eluate (180–250 mM); lane 6, overloaded sample from lane 5. The arrow indicates the position of His-tagged HlyB NBD.

Figure 2. ATPase activity of the purified ABC domain of HlyB

ATPase activity as a function of different salt concentrations, using the enzyme-coupled assay at a protein concentration of 8 μM. Data within 10% deviation of ATPase activity, at 200 mM NaCl, were analysed by the Hill equation (eqn 2, solid line) and the Michaelis–Menten equation (eqn 1, broken line).

Figure 3. Effect of salt concentration on ATPase activity and the fixation of 8-azido-[γ-³²P]ATP by the HlyB ABC ATPase domain

(A) ATPase activity of the purified ABC ATPase (1 μM) at different salt concentrations. The rate of hydrolysis was measured by following the release of P_i for 20 min at 25 °C. (B) Autoradiogram after SDS/PAGE showing the effect of salt on binding 8-azido-[γ-³²P]ATP.

Figure 4. Reciprocal relationship between Trp⁵⁴⁰ fluorescence and ATPase activity of the HlyB ABC ATPase domain with increasing salt concentration

Maximal fluorescence intensity (▲) and ATPase activity, normalized according to Figure 3 (■), were plotted against salt concentration. The lines were fitted to a standard mono-exponential model for dissociation with offset and association respectively.

Figure 5. Vanadate inhibition of the ATPase activity of the HlyB NBD

Experiments were performed in 10 mM Tris/HCl, pH 8.0, and 10 mM KCl with 2 mM ATP, and data were analysed according to eqn (4).

Figure 6. Size-exclusion chromatography of the HlyB NBD

Purified ABC ATPase was eluted from a Superdex HR 75 column with 10 mM Tris/HCl, pH 8.0, and 100 mM KCl, in the absence (profile 1) or presence (profile 2) of non-labelled 8-azido-ATP. For convenience, the two profiles were superimposed after two separate experiments injecting 1.5 mg and 340 μg of protein respectively; however, identical results were obtained with equal 1.5 mg injections. The monomer (28 kDa) and presumed dimer (56 kDa) of His-tagged HlyB ABC are indicated. MM, molecular mass.