The prosegment–subtilisin BPN′ complex: crystal structure of a specific ‘foldase’

Background: The folding of the bacterial protease subtilisin BPN′ (SBT) is dependent on its 77-residue prosegment, which is then autocatalytically removed to give the mature enzyme. Mature subtilisin represents a class of proteins that lacks an efficient folding pathway. Refolding of mature SBT is extremely slow unless catalyzed by the independently expressed prosegment, leading to a bimolecular complex. Results We report the crystal structure at 2.0 a resolution of the prosegment–SBT complex and consider its implications for prosubtilisin BPN′ maturation and folding catalysis. The prosegment forms a compact domain that binds SBT through an extensive interface involving the enzyme's two parallel surface helices (residues 104–116 and 133–144), supplying negatively charged caps to the N termini of these helices. The prosegment C terminus binds in the enzyme active site in a product-like manner, with Tyr77 in the P1 binding pocket. Conclusion The structure of the complex supports a unimolecular mechanism for prosubtilisin cleavage, involving a 25 a rearrangement of the SBT N terminus in a late folding step. A mechanism of folding catalysis in which the two helices and their connecting β strand form a prosegment-stabilized folding nucleus is proposed. While this putative nucleus is stabilized by prosegment binding, the N-terminal and C-terminal subdomains of SBT could fold by propagation.