An intrinsic degradation tag on the ClpA C-terminus regulates the balance of ClpAP complexes with different substrate specificity.

ATP-dependent protein degradation in bacteria is carried out by barrel-shaped proteases architecturally related to the proteasome. In Escherichia coli, ClpP interacts with two alternative ATPases, ClpA or ClpX, to form active protease complexes. ClpAP and ClpXP show different but overlapping substrate specificities. ClpXP is considered the primary recipient of ssrA-tagged substrates while ClpAP in complex with ClpS processes N-end rule substrates. Notably, in its free form, but not in complex with ClpS, ClpAP also degrades ssrA-tagged substrates and its own chaperone component, ClpA. To reveal the mechanism of ClpAP-mediated ClpA degradation, termed autodegradation, and its possible role in regulating ClpAP levels, we dissected ClpA to show that the flexible C-terminus of the second AAA module serves as the degradation signal. We demonstrate that ClpA becomes largely resistant to autodegradation in the absence of its C-terminus and, conversely, transfer of the last 11 residues of ClpA to the C-terminus of green fluorescent protein (GFP) renders GFP a substrate of ClpAP. This autodegradation tag bears similarity to the ssrA-tag in its degradation behavior, displaying similar catalytic turnover rates when coupled to GFP but a twofold lower apparent affinity constant compared to ssrA-tagged GFP. We show that, in analogy to the prevention of ssrA-mediated recognition, the adaptor ClpS inhibits autodegradation by a specificity switch as opposed to direct masking of the degradation signal. Our results demonstrate that in the presence of ssrA-tagged substrates, ClpA autodegradation will be competitively reduced. This simple mechanism allows for dynamic reallocation of free ClpAP versus ClpAPS in response to the presence of ssrA-tagged substrates.