Disabling the folding catalyst is the last critical step in α‐lytic protease folding

Alpha-Lytic protease (alphaLP) is an extracellular bacterial pro-protease marked by extraordinary conformational rigidity and a highly cooperative barrier to unfolding. Although these properties successfully limit its proteolytic destruction, thereby extending the functional lifetime of the protease, they come at the expense of foldability (t(1/2) = 1800 yr) and thermodynamic stability (native alphaLP is less stable than the unfolded species). Efficient folding has required the coevolution of a large N-terminal pro region (Pro) that rapidly catalyzes alphaLP folding (t(1/2) = 23 sec) and shifts the thermodynamic equilibrium in favor of folded protease through tight native-state binding. Release of active alphaLP from this stabilizing, but strongly inhibitory, complex requires the proteolytic destruction of Pro. alphaLP is capable of initiating Pro degradation via cleavage of a flexible loop within the Pro C-terminal domain. This single cleavage event abolishes Pro catalysis while maintaining strong native-state binding. Thus, the loop acts as an Achilles' heel by which the Pro foldase machinery can be safely dismantled, preventing Pro-catalyzed unfolding, without compromising alphaLP native-state stability. Once the loop is cleaved, Pro is rapidly degraded, releasing active alphaLP.