The extracellular bacterial protease, α-lytic protease (αLP), is synthesized with a large, two-domain pro region (Pro) that catalyzes the folding of the protease to its native conformation. In the absence of its Pro folding catalyst, αLP encounters a very large folding barrier (ΔG = 30 kcal mol-1) that effectively prevents the protease from folding (t1/2 of folding = 1800 years). Although homology data, mutational studies, and structural analysis of the Pro·αLP complex suggested that the Pro C-terminal domain (Pro C-domain) serves as the minimum "foldase" unit responsible for folding catalysis, we find that the Pro N-terminal domain (Pro N-domain) is absolutely required for αLP folding. Detailed kinetic analysis of Pro N-domain point mutants and a complete N-domain deletion reveal that the Pro N-domain both provides direct interactions with αLP that stabilize the folding transition state and confers stability to the Pro C-domain. The Pro N- and C-domains make conflicting demands upon native αLP binding that are alleviated in the optimized interface of the folding transition state complex. From these studies, it appears that the extremely high αLP folding barrier necessitates the presence of both the Pro domains; however, αLP homologues with less demanding folding barriers may not require both domains, thus possibly explaining the wide variation in the pro region size of related pro-proteases.
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