Protein binding and unfolding by the chaperone ClpA and degradation by the protease ClpAP.
ClpA, a bacterial member of the Clp/Hsp100 chaperone family, is an ATP-dependent molecular chaperone and the regulatory component of the ATP-dependent ClpAP protease. To study the mechanism of binding and unfolding of proteins by ClpA and translocation to ClpP, we used as a model substrate a fusion protein that joined the ClpA recognition signal from RepA to green fluorescent protein (GFP). ClpAP degrades the fusion protein in vivo and in vitro. The substrate binds specifically to ClpA in a reaction requiring ATP binding but not hydrolysis. Binding alone is not sufficient to destabilize the native structure of the GFP portion of the fusion protein. Upon ATP hydrolysis the GFP fusion protein is unfolded, and the unfolded intermediate can be sequestered by ClpA if a nonhydrolyzable analog is added to displace ATP. ATP is required for release. We found that although ClpA is unable to recognize native proteins lacking recognition signals, including GFP and rhodanese, it interacts with those same proteins when they are unfolded. Unfolded GFP is held in a nonnative conformation while associated with ClpA and its release requires ATP hydrolysis. Degradation of unfolded untagged proteins by ClpAP requires ATP even though the initial ATP-dependent unfolding reaction is bypassed. These results suggest that there are two ATP-requiring steps: an initial protein unfolding step followed by translocation of the unfolded protein to ClpP or in some cases release from the complex.
Laboratory of Molecular Biology and Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J R
JR
Hoskins
S K
SK
Singh
M R
MR
Maurizi
S
S
Wickner
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D049071:Endopeptidase Clp; D049452:Green Fluorescent Proteins; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases