Dynamics of substrate denaturation and translocation by the ClpXP degradation machine.
ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.
Kim
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YI
Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge 02139, USA.
Burton
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RE
Burton
B M
BM
Sauer
R T
RT
Baker
T A
TA
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D049071:Endopeptidase Clp; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008962:Models, Theoretical; D018832:Molecular Chaperones / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D009842:Oligopeptides; D049452:Green Fluorescent Proteins; 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