Both ATPase domains of ClpA are critical for processing of stable protein structures.
ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.
Kress
Wolfgang
W
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
Mutschler
Hannes
H
Weber-Ban
Eilika
E
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D002352:Carrier Proteins; C454820:ClpS protein, E coli; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; C483568:ClpA protease, E coli; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases