Functional domains of the ClpA and ClpX molecular chaperones identified bylimited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that ea ch interact with the protease, ClpP, to promote specific protein degradatio n. We have used limited proteolysis and deletion analysis to probe the conf ormations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gammaS binding stabilized ClpA and ClpX such that that cleavage by lys ylendopeptidase C occurred at only two sites. Both proteins were cleaved wi thin in a loop preceding an a-helix-rich C-terminal domain. Although the lo op varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(FIL). Binding of ClpP blocked this cleavag e, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in inte ractions with ClpP. ClpA was also cut at a site near the junction of the tw o ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other s ites. The N-terminal domain of ClpX dissociated upon cleavage, and the rema ining ClpX DeltaN remained as a hexamer, associated with ClpP, and expresse d ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA l acking its N-terminal 153 amino acids also formed a hexamer, associated wit h ClpP, and expressed these activities. We propose that the N-terminal doma ins of ClpX and ClpA lie on the outside ring surface of the holoenzyme comp lexes where they contribute to substrate binding or perform a gating functi on affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.