Document Detail


Roles of the N-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease.
MedLine Citation:
PMID:  16207718     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
J?rg Hinnerwisch; Brian G Reid; Wayne A Fenton; Arthur L Horwich
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2005-10-05
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  280     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2005 Dec 
Date Detail:
Created Date:  2005-12-05     Completed Date:  2006-03-06     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  40838-44     Citation Subset:  IM    
Affiliation:
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
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MeSH Terms
Descriptor/Qualifier:
Allosteric Site
Binding Sites
Biological Transport
Endopeptidase Clp / chemistry*,  genetics,  metabolism*
Fluorescence Resonance Energy Transfer
Gene Expression
Green Fluorescent Proteins / genetics,  metabolism
Luciferases / metabolism
Mutagenesis
Peptide Fragments / metabolism
Protein Folding
RNA, Bacterial
Recombinant Fusion Proteins / metabolism
Structure-Activity Relationship
Substrate Specificity
Chemical
Reg. No./Substance:
0/Peptide Fragments; 0/RNA, Bacterial; 0/Recombinant Fusion Proteins; 0/tmRNA; 147336-22-9/Green Fluorescent Proteins; EC 1.13.12.-/Luciferases; EC 3.4.21.92/Endopeptidase Clp

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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