Characterization of a specificity factor for an AAA+ ATPase: assembly of SspB dimers with ssrA-tagged proteins and the ClpX hexamer.
SspB, a specificity factor for the ATP-dependent ClpXP protease, stimulates proteolysis of protein substrates bearing the ssrA degradation tag. The SspB protein is shown here to form a stable homodimer with two independent binding sites for ssrA-tagged proteins or peptides. SspB by itself binds to ClpX and stimulates the ATPase activity of this enzyme. In the presence of ATPgammaS, a ternary complex of SspB, GFP-ssrA, and the ClpX ATPase was sufficiently stable to isolate by gel-filtration or ion-exchange chromatography. This complex consists of one SspB dimer, two molecules of GFP-ssrA, and one ClpX hexamer. SspB dimers do not commit bound substrates to ClpXP degradation but increase the affinity and cooperativity of binding of ssrA-tagged substrates to ClpX, facilitating enhanced degradation at low substrate concentrations.
Wah
David A
DA
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Levchenko
Igor
I
Baker
Tania A
TA
Sauer
Robert T
RT
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D039223:Cleavage And Polyadenylation Specificity Factor / Q000378:metabolism*; D019281:Dimerization; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*
D002352:Carrier Proteins; D039223:Cleavage And Polyadenylation Specificity Factor; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D011506:Proteins; C477700:sspB protein, E coli; D049452:Green Fluorescent Proteins; 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