title: Human mitochondrial ClpP is a stable heptamer that assembles into a tetradecamer in the presence of ClpX.
abstract: The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.
authors.affiliation: Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Sung Gyun
authors.initials: SG
authors.lastname: Kang
authors.forename: Mariana N
authors.initials: MN
authors.lastname: Dimitrova
authors.forename: Joaquin
authors.initials: J
authors.lastname: Ortega
authors.forename: Ann
authors.initials: A
authors.lastname: Ginsburg
authors.forename: Michael R
authors.initials: MR
authors.lastname: Maurizi
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D049071:Endopeptidase Clp / Q000737:chemistry*; D005976:Glutaral / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008099:Liver / Q000378:metabolism; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014461:Ultracentrifugation
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D014357:Trypsin; D049071:Endopeptidase Clp; D003545:Cysteine; D005976:Glutaral