title: Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX.
abstract: The hexameric AAA+ ring of Escherichia coli ClpX, an ATP-dependent machine for protein unfolding and translocation, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+-ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U↔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+-ring activity.
authors: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors: Benjamin M
authors: BM
authors: Stinson
authors: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors: Vladimir
authors: V
authors: Baytshtok
authors: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors: Karl R
authors: KR
authors: Schmitz
authors: 1] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors: Tania A
authors: TA
authors: Baker
authors: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors: Robert T
authors: RT
authors: Sauer
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D049071:Endopeptidase Clp / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000378:metabolism; D058767:Protein Unfolding
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D009711:Nucleotides; D021122:Protein Subunits; D000255:Adenosine Triphosphate; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities