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ATP-Dependent Conformational Changes and Translocation of Substrates in Clpap Protease as Revealed by Cryo-Electron Microscopy

Published online by Cambridge University Press:  02 July 2020

Takashi Ishikawa ,
Fabienne Beuron ,
Martin Kessel ,
Sue Wickner ,
Michael R. Maurizi  and
Alasdair C. Steven
Takashi Ishikawa
Affiliation:
Laboratory of Structural Biology, NIAMS; National Institutes of Health, Bethesda, MD20892, USA
Fabienne Beuron
Affiliation:
Laboratory of Structural Biology, NIAMS; National Institutes of Health, Bethesda, MD20892, USA
Martin Kessel
Affiliation:
Laboratory of Structural Biology, NIAMS; National Institutes of Health, Bethesda, MD20892, USA
Sue Wickner
Affiliation:
Cell Biology, National Institutes of Health, Bethesda, MD20892, USA
Michael R. Maurizi
Affiliation:
Molecular Biology, NCI, National Institutes of Health, Bethesda, MD20892, USA
Alasdair C. Steven
Affiliation:
Laboratory of Structural Biology, NIAMS; National Institutes of Health, Bethesda, MD20892, USA
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Extract

ClpAP, an ATP-dependent protease of E. coli, recognizes and unfolds protein substrates via ClpA, its chaperonelike ATPase component, and digests them in ClpP, its protease component . ClpA forms hexameric rings with a two-layered structure, and stacks axially on either face of the double heptameric rings of ClpP. Protein substrates can bind to ClpAP in the presence of ATPγS, which is not hydrolyzed by ClpA, but are not degraded unless ATP is added. This property makes it possible to synchronize degradation in vitro by forming enzymesubstrate complexes in the presence of ATPγS and then adding ATP to trigger subsequent steps. We have used image averaging of electron micrographs of frozen hydrated and negatively stained specimens to characterize interactions of ClpA and ClpAP complexes with the model substrate, bacteriophage P1 protein, RepA.

Type
Electron Cryomicroscopy of Macromolecules
Information
Microscopy and Microanalysis , Volume 6 , Issue S2: Proceedings: Microscopy & Microanalysis 2000, Microscopy Society of America 58th Annual Meeting, Microbeam Analysis Society 34th Annual Meeting, Microscopical Society of Canada/Societe de Microscopie de Canada 27th Annual Meeting, Philadelphia, Pennsylvania August 13-17, 2000 , August 2000 , pp. 260 - 261
Copyright
Copyright © Microscopy Society of America

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References

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