An open axial channel of the <scp>AAA ClpXP</scp> protease enhances degradation of specific classes of protein substrates

ClpXP and other AAA proteases maintain proteostasis and regulate cellular functions by degrading misfolded, incomplete, or regulatory proteins. ClpX recognizes substrates via unstructured degron sequences, typically located at the N- or C-terminus. Although five classes of degrons are known, only recognition of the ssrA tag, a C-motif-1 degron, is well understood. The ssrA tag initially binds to a conformation of hexameric ClpX in which the axial channel is closed by a pore-2 loop, with subsequent channel opening allowing translocation into and degradation by ClpP. A ClpX variant with a pore-2 loop deletion (ΔNPS) favors the open conformation and exhibits weaker binding to ssrA-tagged substrates. Here, using model substrates representing each of the five known degron classes, we show that ΔNPS ClpXP degrades low micromolar concentrations of N-motif-1, -2, -3, and C-motif-2 substrates more effectively than wild-type ClpXP. Cryo-EM analysis of wild-type ClpXP bound to an N-motif-1 substrate reveals degron engagement within an open axial channel. Our results support a model in which the open- and closed-channel conformations of ClpXP differentially enhance recognition of distinct degron classes: the open channel facilitates degradation of many natural substrates, whereas the closed channel promotes efficient recognition and degradation of ssrA-tagged proteins. We propose that the conformational equilibrium between these two states tunes ClpXP activity to balance broad substrate recognition with specificity, allowing cells to meet dynamic proteolytic demands and minimize off-target degradation.