Protein binding and unfolding by the chaperone ClpA and degradation by theprotease ClpAP

ClpA, a bacterial member of the Clp/Hsp100 chaperone family, is an ATP-depe ndent molecular chaperone and the regulatory component of the ATP-dependent ClpAP protease, To study the mechanism of binding and unfolding of protein s 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 fluoresc ent protein (GFP). ClpAP degrades the fusion protein in vivo and in vitro. The substrate binds specifically to ClpA in a reaction requiring ATP bindin g but not hydrolysis. Binding alone is not sufficient to destabilize the na tive structure of the GFP portion of the fusion protein. Upon ATP hydrolysi s the GFP fusion protein is unfolded, and the unfolded intermediate can be sequestered by ClpA if a nonhydrolyzable analog is added to displace ATP. A TP is required for release. We found that although ClpA is unable to recogn ize native proteins lacking recognition signals, including GFP and rhodanes e, it interacts with those same proteins when they are unfolded. Unfolded G FP is held in a nonnative conformation while associated with ClpA and its r elease requires ATP hydrolysis. Degradation of unfolded untagged proteins b y ClpAP requires ATP even though the initial ATP-dependent unfolding reacti on is bypassed. These results suggest that there are two ATP-requiring step s: an initial protein unfolding step followed by translocation of the unfol ded protein to ClpP or in some cases release from the complex.