Topologically complex knotted proteins are processed differentially by ATP ‐dependent proteases ClpXP and ClpAP

Abstract Knotted proteins can present a distinct topological and mechanical challenge to the AAA+ cellular degradation machineries, determined by the relative size of the knot and the pore dimensions of these enzymes. In this study, we investigated the degradation mechanism of knotted proteins of different topological complexities by two E. coli. AAA+ proteases ClpXP and ClpAP. While ClpXP efficiently degraded all knotted substrates, regardless of their topology, ClpAP exhibited a striking functional divergence. Despite its robust double‐ring ATPase architecture, ClpAP failed to degrade the 4 1 ‐knotted miRFP and degraded the 3 1 ‐knotted CAIX and more complex 5 2 ‐knotted UCHL1 slower than ClpXP. We have demonstrated that this disparity is not dictated merely by absolute knot complexity, but is critically governed by the length of the knot tail, which determines whether a knot can spontaneously unthread or is forced to tighten during the translocation. Furthermore, based on the slower processing of a sterically constrained covalent dimeric substrate Arc‐ssrA, we propose that ClpAP's hindered processing of knotted substrates stems from its smaller axial pore. Overall, we propose a unified physical model in which the successful degradation of a knotted protein relies on a delicate interplay among knot compaction, tail‐mediated unthreading, and the architectural permissiveness of the unfoldase pore.