title: Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP.
abstract: Binding and internalization of a protein substrate by E. coli ClpXP was investigated by electron microscopy. In sideviews of ATP gamma S-stabilized ClpXP complexes, a narrow axial channel was visible in ClpX, surrounded by protrusions on its distal surface. When substrate lambda O protein was added, extra density attached to this surface. Upon addition of ATP, this density disappeared as lambda O was degraded. When ATP was added to proteolytically inactive ClpXP-lambda O complexes, the extra density transferred to the center of ClpP and remained inside ClpP after separation from ClpX. We propose that substrates of ATP-dependent proteases bind to specific sites on the distal surface of the ATPase, and are subsequently unfolded and translocated into the internal chamber of the protease.
authors.affiliation: Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: J
authors.initials: J
authors.lastname: Ortega
authors.forename: S K
authors.initials: SK
authors.lastname: Singh
authors.forename: T
authors.initials: T
authors.lastname: Ishikawa
authors.forename: M R
authors.initials: MR
authors.lastname: Maurizi
authors.forename: A C
authors.initials: AC
authors.lastname: Steven
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D010582:Bacteriophage lambda; D001665:Binding Sites; D019281:Dimerization; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007091:Image Processing, Computer-Assisted; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D018832:Molecular Chaperones; D011107:Polylysine / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D014764:Viral Proteins / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; C027812:O protein, Bacteriophage lambda; D014764:Viral Proteins; D011107:Polylysine; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities