Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli - Sorbonne Université Access content directly
Journal Articles Biochemical Journal Year : 2005

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

Lutz Schmitt
  • Function : Author
Carsten Horn
  • Function : Author
Kornelia Jumel
  • Function : Author
Mark A. Blight
  • Function : Author
I. Barry Holland
  • Function : Author

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 μM, 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.

Dates and versions

hal-03943728 , version 1 (17-01-2023)

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Houssain Benabdelhak, Lutz Schmitt, Carsten Horn, Kornelia Jumel, Mark A. Blight, et al.. Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli. Biochemical Journal, 2005, 386 (3), pp.489-495. ⟨10.1042/BJ20041282⟩. ⟨hal-03943728⟩
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