Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli. HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB.
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A schematic representation of the potential effect (in low intracellular Ca2+ conditions) of the Ca2+-binding, EF-hand-containing cytoskeletal protein EFhd2 on kinesin-mediated transport and actin dynamics in presynaptic compartments. For a detailed overview of the links between EFhd2 and neurodegeneration, see the review by Mielenz and Gunn-Moore in this issue (Volume 473, Issue 16, pages 2429–2437). - PDF Icon PDF LinkFront Matter
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Research Article|
August 11 2016
Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli
Sven Reimann;
Sven Reimann
*Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
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Gereon Poschmann;
Gereon Poschmann
†Molecular Proteomics Laboratory, Biomedizinisches Forschungszentrum (BMFZ), Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
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Kerstin Kanonenberg;
Kerstin Kanonenberg
*Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
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Kai Stühler;
Kai Stühler
†Molecular Proteomics Laboratory, Biomedizinisches Forschungszentrum (BMFZ), Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
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Sander H.J. Smits;
Sander H.J. Smits
*Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
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Lutz Schmitt
Lutz Schmitt
1
*Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
‡Center of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
1To whom correspondence should be addressed (email Lutz.Schmitt@hhu.de).
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Publisher: Portland Press Ltd
Received:
February 23 2016
Revision Received:
May 23 2016
Accepted:
June 07 2016
Accepted Manuscript online:
June 08 2016
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society
2016
Biochem J (2016) 473 (16): 2471–2483.
Article history
Received:
February 23 2016
Revision Received:
May 23 2016
Accepted:
June 07 2016
Accepted Manuscript online:
June 08 2016
Citation
Sven Reimann, Gereon Poschmann, Kerstin Kanonenberg, Kai Stühler, Sander H.J. Smits, Lutz Schmitt; Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli. Biochem J 15 August 2016; 473 (16): 2471–2483. doi: https://doi.org/10.1042/BCJ20160154
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