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

• Published in: Cell Vol. 153; no. 3; pp. 628 - 639
• Main Authors: Stinson, Benjamin M., Nager, Andrew R., Glynn, Steven E., Schmitz, Karl R., Baker, Tania A., Sauer, Robert T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.04.2013
• 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
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2013.03.029

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.