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.
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Research Article|
March 08 2005
Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli
Houssain BENABDELHAK;
Houssain BENABDELHAK
1
*Institut de Génétique et Microbiologie, Bâtiment 409, Université Paris-Sud, 91405 Orsay Cedex, France
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Lutz SCHMITT;
Lutz SCHMITT
†Institute of Biochemistry, Biocenter N210, Johann Wolfgang Goethe University Frankfurt, Marie-Curie Strasse 9, 60439 Frankfurt, Germany
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Carsten HORN;
Carsten HORN
†Institute of Biochemistry, Biocenter N210, Johann Wolfgang Goethe University Frankfurt, Marie-Curie Strasse 9, 60439 Frankfurt, Germany
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Kornelia JUMEL;
Kornelia JUMEL
‡School of Biosciences, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Leics. LE12 5RD, U.K.
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Mark A. BLIGHT;
Mark A. BLIGHT
*Institut de Génétique et Microbiologie, Bâtiment 409, Université Paris-Sud, 91405 Orsay Cedex, France
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I. Barry HOLLAND
I. Barry HOLLAND
2
*Institut de Génétique et Microbiologie, Bâtiment 409, Université Paris-Sud, 91405 Orsay Cedex, France
2To whom correspondence should be addressed (email barry.holland@igmors.u-psud.fr).
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Publisher: Portland Press Ltd
Received:
July 30 2004
Revision Received:
December 16 2004
Accepted:
January 07 2005
Accepted Manuscript online:
January 07 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 386 (3): 489–495.
Article history
Received:
July 30 2004
Revision Received:
December 16 2004
Accepted:
January 07 2005
Accepted Manuscript online:
January 07 2005
Citation
Houssain BENABDELHAK, Lutz SCHMITT, Carsten HORN, Kornelia JUMEL, Mark A. BLIGHT, I. Barry HOLLAND; Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli. Biochem J 15 March 2005; 386 (3): 489–495. doi: https://doi.org/10.1042/BJ20041282
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