title: Roles of the N-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease.
abstract: The hexameric cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding and translocation of recognized substrate proteins into the coaxially associated serine protease ClpP. Each subunit of ClpA is composed of an N-terminal domain of approximately 150 amino acids at the top of the cylinder followed by two AAA+ domains. In earlier studies, deletion of the N-domain was shown to have no effect on the rate of unfolding of substrate proteins bearing a C-terminal ssrA tag, but it did reduce the rate of degradation of these proteins (Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Protein Sci. 10, 551-559; Singh, S. K., Rozycki, J., Ortega, J., Ishikawa, T., Lo, J., Steven, A. C., and Maurizi, M. R. (2001) J. Biol. Chem. 276, 29420-29429). Here we demonstrate, using both fluorescence resonance energy transfer to measure the arrival of substrate at ClpP and competition between wild-type and an inactive mutant form of ClpP, that this effect on degradation is caused by diminished stability of the ClpA-ClpP complex during translocation and proteolysis, effectively disrupting the targeting of unfolded substrates to the protease. We have also examined two larger ssrA-tagged substrates, CFP-GFP-ssrA and luciferase-ssrA, and observed different behaviors. CFP-GFP-ssrA is not efficiently unfolded by the truncated chaperone whereas luciferase-ssrA is, suggesting that the former requires interaction with the N-domains, likely via the body of the protein, to stabilize its binding. Thus, the N-domains play a key allosteric role in complex formation with ClpP and may also have a critical role in recognizing certain tag elements and binding some substrate proteins.
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: Jörg
authors.initials: J
authors.lastname: Hinnerwisch
authors.forename: Brian G
authors.initials: BG
authors.lastname: Reid
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Fenton
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Horwich
mesh_terms: D000495:Allosteric Site; D001665:Binding Sites; D001692:Biological Transport; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism; D017510:Protein Folding; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; C096222:tmRNA; D049452:Green Fluorescent Proteins; D008156:Luciferases; D049071:Endopeptidase Clp