Voedts, H.; Nguyen, P. C.; Nguyen, V.-S.; Leverrier, P.; Iorga, B. I.; Cho, S.-H.; Remaut, H.; Collet, J.-F.

Proteases must be tightly regulated to prevent uncontrolled degradation, yet the mechanisms ensuring such control remain poorly understood. Members of the widespread M48 metalloprotease family are kept inactive by an autoinhibitory plug that blocks catalytic water activation, but how this plug is released was unknown. Here, using genetic, biochemical and cryo-EM approaches, we discover the activation mechanism of BepA, a quality-control protease that preserves outer membrane integrity by surveilling the {beta}-barrel assembly machinery (BAM) in Gram-negative bacteria. Our cryo-EM analysis of BepA engaged with a stalled BAM-substrate assembly complex revealed that a flexible, unstructured 6-lid covering the active site in the latent protein functions as a molecular harpoon, inserting into the outer membrane when a substrate stalls at BAM and thereby docking BepA at the complex. This membrane anchoring promotes displacement of the autoinhibitory plug and unlocks protease activity precisely where and when it is needed. Thus, a dual enzyme activation mechanism is coupled to membrane association under stress, ensuring that BepA remains inactive until properly localized. Our findings reveal how membranes themselves can license protease activation, a principle that may extend beyond M48 metalloproteases.