Structural basis for autoinhibition and self-activation in the Pseudomonas aeruginosa virulence factor protease IV (PIV)

Abstract

Secreted proteases enable Pseudomonas aeruginosa to damage host tissues and evade host immune defences, but the molecular basis by which protease IV (PIV) is maintained as an inactive precursor remains unclear. Here we determined a 2.17 A crystal structure of catalytically inactive PIVS409A. The precursor comprises an N-terminal Cap region, a CUB domain and a C-terminal trypsin-like protease domain. The Cap region forms an extended clamp across the precursor, and residues at the Cap-CUB junction lie above the catalytic cleft. Structural comparison with a modelled peptide substrate indicates that the Cap does not engage the substrate-recognition pockets as a pseudosubstrate; instead, it sterically prevents access to the S1 pocket, oxyanion hole and catalytic triad. Wild-type PIV undergoes progressive processing to a 27.6-kDa mature species, whereas the S409A variant remains resistant to activation by exogenous mature enzyme, supporting an intramolecular initiating step. Mature PIV is weakly inhibited by an ornithine-containing peptide mimicking the Cap domain, is not inhibited by phenylmethylsulfonyl fluoride and is slowly irreversibly inhibited by TLCK, demonstrating unique characteristics in comparison to other proteases from the same family. Finally, mature PIV increases the activity of aminopeptidase PaAP, likely through maturation of this enzyme, as part of a protease activation cascade. These findings define the architecture of the PIV precursor and provide a structural framework for understanding its activation and selective autoinhibition.