title: Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle.
abstract: The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Alvaro J
authors.initials: AJ
authors.lastname: Amor
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Karl R
authors.initials: KR
authors.lastname: Schmitz
authors.affiliation: Department of Chemistry, Brown University, Providence, RI 02912.
authors.forename: Jason K
authors.initials: JK
authors.lastname: Sello
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.|Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D015374:Biosensing Techniques; D047630:Depsipeptides / Q000737:chemistry; D049071:Endopeptidase Clp / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry*; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D019809:Streptavidin / Q000737:chemistry
chemical_list: C000622571:ADEP-2B depsipeptide; D047630:Depsipeptides; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D019809:Streptavidin; C483572:ClpP protease, E coli; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities