Dynamics of substrate denaturation and translocation by the ClpXP degradation machine

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp /Hsp100 family of remodeling enzymes, and the clop peptidase. Here, ClpX an d ClpXP are shown to catalyze denaturation of GFP modified with an ssrA deg radation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release o f denatured GFP-ssrA from ClpP in a reaction that requires ATP and addition al substrate. Kinetic experiments reveal that multiple reaction steps requi re collaboration between ClpX and ClpP and that denaturation is the rate-de termining step in degradation. These insights into the mechanism of ClpXP e xplain how it executes efficient degradation in a manner that is highly spe cific for tagged proteins, irrespective of their intrinsic stabilities.