Characterization of a trap mutant of the AAA+ chaperone ClpB.
The AAA+ protein ClpB mediates the solubilization of protein aggregates in cooperation with the DnaK chaperone system (KJE). The order of action of ClpB and KJE on aggregated proteins is unknown. We describe a ClpB variant with mutational alterations in the Walker B motif of both AAA domains (E279A/E678A), which binds but does not hydrolyze ATP. This variant associates in vitro and in vivo in a stable manner with protein substrates, demonstrating direct interaction of ClpB with protein aggregates for the first time. Substrate interaction is strictly dependent on ATP binding to both AAA domains of ClpB. The unique substrate binding properties of the double Walker B variant allowed to dissect the order of ClpB and DnaK action during disaggregation reactions. ClpB-E279A/E678A outcompetes the DnaK system for binding to the model substrate TrfA and inhibits the dissociation of small protein aggregates by DnaK only, indicating that ClpB acts prior to DnaK on protein substrates.
Weibezahn
Jimena
J
ZMBH, Universität Heidelberg, Im NeuenheimerFeld 282, Heidelberg D-69120, Germany.
Schlieker
Christian
C
Bukau
Bernd
B
Mogk
Axel
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000378:metabolism; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics*; D006868:Hydrolysis; D015151:Immunoblotting; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013997:Time Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C067405:TrfA protein, E coli; D000255:Adenosine Triphosphate; D008156:Luciferases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; C069775:ClpB protein, E coli