Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine

ClpXP is an ATP-dependent protease that denatures native proteins and trans locates the denatured polypeptide into an interior peptidase chamber for de gradation, To address the mechanism of these processes. Arc repressor varia nts with dramatically different stabilities and unfolding half-lives varyin g from months to seconds were targeted to ClpXP by addition of the ssrA deg radation tag. Remarkably, ClpXP degraded each variant at a very similar rat e and hydrolyzed similar to 150 molecules of ATP for each molecule of subst rate degraded. The hyperstable substrates did, however, slow the ClpXP ATPa se cycle. These results confirm that ClpXP uses an active mechanism to dena ture its substrates, probably one that applies mechanical force to the nati ve structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficien tly, even when just one subunit contained an ssrA tag. This result indicate s that the pore through which denatured proteins enter the proteolytic cham ber must be large enough to accommodate simultaneous passage of two or thre e polypeptide chains.