title: Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine.
abstract: 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.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Stinson
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Nager
authors.forename: Steven E
authors.initials: SE
authors.lastname: Glynn
authors.forename: Karl R
authors.initials: KR
authors.lastname: Schmitz
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D009711:Nucleotides; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities