STRUCTURE OF ALPHA-LYTIC PROTEASE COMPLEXED WITH ITS PRO REGION

While the majority of proteins fold rapidly and spontaneously to their native states, the extracellular bacterial protease alpha-lytic prote ase (alpha LP) has a t(1/2) for folding of similar to 2,000 years, cor responding to a folding barrier of 30 kcal mol(-1). alpha LP is synthe sized as a pro-enzyme where its pro region (Pro) acts as a foldase to stabilize the transition state for the folding reaction. Pro also func tions as a potent folding catalyst when supplied as a separate polypep tide chain, accelerating the rate of alpha LP folding by a factor of 3 x 10(9). In the absence of Pro, alpha LP folds only partially to a st able molten globule-like intermediate state. Addition of Pro to this i ntermediate leads to rapid formation of native alpha LP. Here we repor t the crystal structures of Pro and of the non-covalent inhibitory com plex between Pro and native alpha LP. The C-shaped Pro surrounds the C -terminal beta-barrel domain of the folded protease, forming a large c omplementary interface. Regions of extensive hydration in the interfac e explain how Pro binds tightly to the native state, yet even more tig htly to the folding transition state. Based on structural and function al data we propose that a specific structural element in alpha LP is l argely responsible for the folding barrier and suggest how Pro can ove rcome this barrier.