The crystal structure of a Cys25 -> Ala mutant of human procathepsin S elucidates enzyme-prosequence interactions.

The crystal structure of the active-site mutant Cys25 --> Ala of glycosylated human procathepsin S is reported. It was determined by molecular replacement and refined to 2.1 Angstrom resolution, with an R-factor of 0.198. The overall structure is very similar to other cathepsin L-like zymogens of the C1A clan. The peptidase unit comprises two globular domains, and a small third domain is formed by the N-terminal part of the prosequence. It is anchored to the prosegment binding loop of the enzyme. Prosegment residues beyond the prodomain dock to the substrate binding cleft in a nonproductive orientation. Structural comparison with published data for mature cathepsin S revealed that procathepsin S residues Phe146, Phe70, and Phe211 adopt different orientations. Being part of the S1' and S2 pockets, they may contribute to the selectivity of ligand binding. Regarding the prosequence, length, orientation and anchoring of helix alpha3p differ from related zymogens, thereby possibly contributing to the specificity of propeptide-enzyme interaction in the papain family. The discussion focuses on the functional importance of the most conserved residues in the prosequence for structural integrity, inhibition and folding assistance, considering scanning mutagenesis data published for procathepsin S and for its isolated propeptide.