PMID:15037252
| Citation |
Ortega, J, Lee, HS, Maurizi, MR and Steven, AC ClpA and ClpX ATPases bind simultaneously to opposite ends of ClpP peptidase to form active hybrid complexes. J. Struct. Biol. 146:217-26 |
|---|---|
| Abstract |
The Escherichia coli ATP-dependent ClpAP and ClpXP proteases are composed of a single proteolytic component, ClpP, complexed with either of the two related chaperones, ClpA or ClpX. ClpXP and ClpAP complexes interact with different specific substrates and catalyze ATP-dependent protein unfolding and degradation. In vitro in the presence of ATP or ATPgammaS, ClpA and ClpX form homomeric rings of six subunits, which bind to one or both ends of the double heptameric rings of ClpP. We have observed that, when equimolar amounts of ClpA and ClpX hexamers are added to ClpP in vitro in the presence of ATP or ATPgammaS, hybrid complexes in which ClpX and ClpA are bound to opposite ends of the same ClpP are readily formed. The distribution of homomeric and heteromeric complexes was consistent with random binding of ClpA and ClpX to the ends of ClpP. Direct demonstration of the functionality of the heteromeric complexes was obtained by electron microscopy, which allowed us to visualize substrate translocation into proteolytically inactive ClpP chambers. Starting with hybrid complexes to which protein substrates specific to ClpX or ClpA were bound, translocation of both types of substrates was shown to occur without significant redistribution of ClpA or ClpX. The stoichiometric ratios of the ClpA, ClpX, and ClpP oligomeric complexes in vivo are consistent with the predominance of heteromeric complexes in growing cells. Thus, ClpXAP is a bifunctional protease whose two ends can independently target different classes of substrates. |
| Links |
PubMed Online version:10.1016/j.jsb.2003.11.023 |
| Keywords |
Adenosine Triphosphatases; Binding Sites; Endopeptidase Clp; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Macromolecular Substances; Microscopy, Electron; Molecular Chaperones/chemistry; Protein Binding; Substrate Specificity |
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