Nucleotide Binding and Conformational Switching in the Hexameric Ring of a AAA+ Machine

ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt u...

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Bibliographic Details
Published in:Cell 2013-04, Vol.153 (3), p.628-639
Main Authors: Stinson, Benjamin M., Nager, Andrew R., Glynn, Steven E., Schmitz, Karl R., Baker, Tania A., Sauer, Robert T.
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - chemistry
Adenosine Triphosphatases - genetics
Adenosine Triphosphatases - metabolism
adenosine triphosphate
Adenosine Triphosphate - metabolism
ATPases Associated with Diverse Cellular Activities
crystal structure
Endopeptidase Clp - chemistry
Endopeptidase Clp - genetics
Endopeptidase Clp - metabolism
energy
Escherichia coli - metabolism
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
fluorescence
hydrolysis
Models, Molecular
Molecular Chaperones - chemistry
Molecular Chaperones - genetics
Molecular Chaperones - metabolism
mutagenesis
Nucleotides - metabolism
Protein Conformation
Protein Folding
Protein Subunits - chemistry
Protein Subunits - genetics
Protein Subunits - metabolism
proteins
Proteolysis
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Summary:ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt unloadable conformations, and each conformational class exhibits substantial variability. Using mutagenesis of individual subunits in covalently tethered hexamers together with fluorescence methods to assay the conformations and nucleotide-binding properties of these subunits, we demonstrate that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work. ATP binding to different classes of subunits initially drives staged allosteric changes, which set the conformation of the ring to allow hydrolysis and linked mechanical steps. Subunit switching between loadable and unloadable conformations subsequently isomerizes or resets the configuration of the nucleotide-loaded ring and is required for mechanical function. [Display omitted] •Structures show nucleotide-loadable and unloadable subunits of AAA+ ClpX ATPase•Assays detects subunit-specific ATP binding and conformational changes•ATP binding drives staged allosteric changes in the ClpX ring•Work requires dynamic interconversion between loadable and unloadable conformations A new assay for AAA+ machines reveals that subunits of the ClpX homohexamer convert between ATP-loadable and unloadable during the functional cycle of ClpX and that ATP binding initiates stepwise allosteric changes in the ClpX ring.
ISSN:0092-8674
1097-4172
DOI:10.1016/j.cell.2013.03.029