Abstact

ClpXP and other AAA+ proteases play central roles in bacterial proteostasis by degrading misfolded and regulatory proteins. In Pseudomonas aeruginosa, ClpXP consists of the ClpX unfoldase and ClpP peptidase, which influence critical adaptive processes contributing to stress resistance. P. aeruginosa (Pa)ClpP1 and (Pa)ClpP2 paralogs assemble into homomeric ((Pa)ClpP1*ClpP1) and heteromeric ((Pa)ClpP1*ClpP2) complexes. (Pa)ClpP2 is only active in the (Pa)ClpP1*ClpP2 heterocomplex. Here, we present a cryo-EM structure of (Pa)ClpX*ClpP1*ClpP2, revealing how (Pa)ClpX binds (Pa)ClpP1, which in turn interacts with (Pa)ClpP2. Comparison of the active heterocomplex with an inactive (Pa)ClpP2 crystal structure shows that (Pa)ClpP1 binding induces conformational changes in (Pa)ClpP2, stabilizing an active catalytic triad. Differences in (Pa)ClpP1 and (Pa)ClpP2 substrate-binding residues and an unstructured ClpP2 N-terminal segment that protrudes into the peptidase chamber likely contribute to distinct peptide-cleavage specificities of (Pa)ClpX*ClpP1*ClpP2 and (Pa)ClpX*ClpP1*ClpP1. Given the role of (Pa)ClpP1*ClpP2 in biofilm formation and virulence, these structural insights may provide a foundation for developing selective inhibitors to combat P. aeruginosa infections.