The hexameric AAA+ (ATPase associated with various cellular activities) chaperone ClpB reactivates protein aggregates in collaboration with the DnaK system. An intriguing aspect of ClpB function is that the active hexamer is unstable and therefore questions how this chaperone uses multiple rounds of ATP hydrolysis to translocate substrates through its central channel. In the present paper, we report the use of biochemical and fluorescence tools to explore ClpB dynamics under different experimental conditions. The analysis of the chaperone activity and the kinetics of subunit exchange between protein hexamers labelled at different protein domains indicates, in contrast with the current view, that (i) ATP favours assembly and ADP dissociation of the hexameric assembly, (ii) subunit exchange kinetics is at least one order of magnitude slower than the ATP hydrolysis rate, (iii) ClpB dynamics and activity are related processes, and (iv) DnaK and substrate proteins regulate the ATPase activity and dynamics of ClpB. These data suggest that ClpB hexamers remain associated during several ATP hydrolysis events required to partially or completely translocate substrates through the protein central channel, and that ClpB dynamics is tuned by DnaK and substrate proteins.
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Research Article|
March 06 2015
ClpB dynamics is driven by its ATPase cycle and regulated by the DnaK system and substrate proteins
Alejandra Aguado;
Alejandra Aguado
*Biophysics Unit, Spanish Science Research Council University of the Basque Country (CSIC-UPV/EHU), and Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain
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José Angel Fernández-Higuero;
José Angel Fernández-Higuero
*Biophysics Unit, Spanish Science Research Council University of the Basque Country (CSIC-UPV/EHU), and Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain
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Yovana Cabrera;
Yovana Cabrera
*Biophysics Unit, Spanish Science Research Council University of the Basque Country (CSIC-UPV/EHU), and Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain
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Fernando Moro;
Fernando Moro
*Biophysics Unit, Spanish Science Research Council University of the Basque Country (CSIC-UPV/EHU), and Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain
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Arturo Muga
Arturo Muga
1
*Biophysics Unit, Spanish Science Research Council University of the Basque Country (CSIC-UPV/EHU), and Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain
1To whom correspondence should be addressed (email arturo.muga@ehu.es).
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Publisher: Portland Press Ltd
Received:
November 12 2014
Revision Received:
December 19 2014
Accepted:
January 05 2015
Accepted Manuscript online:
January 05 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2015 Biochemical Society
2015
Biochem J (2015) 466 (3): 561–570.
Article history
Received:
November 12 2014
Revision Received:
December 19 2014
Accepted:
January 05 2015
Accepted Manuscript online:
January 05 2015
Citation
Alejandra Aguado, José Angel Fernández-Higuero, Yovana Cabrera, Fernando Moro, Arturo Muga; ClpB dynamics is driven by its ATPase cycle and regulated by the DnaK system and substrate proteins. Biochem J 15 March 2015; 466 (3): 561–570. doi: https://doi.org/10.1042/BJ20141390
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