Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at the wrong time and location may be lethal. Two principal mechanisms to control the activity of proteases have been developed during evolution. The first is the co-evolution of endogenous inhibitors, typically occurring in cellular compartments separated from those containing active enzymes. The second is the fact that proteases are synthesized as inactive or less active precursor molecules. They are activated, in some cases, upon an appropriate signal like acidification, Ca(++) -binding or, in other cases, by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize experimental data concerning three functions of propeptides of clan CA family C1 cysteine peptidases (papain family), namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Cysteine peptidases of the CA-C1 family include members from the plant kingdom like papain as well as from the animal kingdom like the lysosomal cathepsins L and B. As it will be shown, the functions are determined by certain structural motifs conserved over millions of years after the evolutionary trails have diverged. The function of propeptides of two other important classes of cysteine peptidases - the calpains, clan CA family C4, and the caspases, clan CD family C 14 - are not considered in this review.
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 Å 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 S19 and S2 pockets, they may contribute to the selectivity of ligand binding. Regarding the prosequence, length, orientation and anchoring of helix a3p 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.
Folding of cathepsin S, like other cathepsin L-like proteases, depends on its proregion. The major part of the proregion forms a small domain distal from the catalytic centre, suggesting function(s) beyond active-site shielding. Using an optimised in vitro trans-refolding assay, we compared reactivation of denatured cathepsin S by the genuine propeptide, wild-type and ten selected mutants. Including structural data and binding constants, we identified the prodomain core and the hairpin region to be important for the foldase function.
Proteins that are unstable or poorly soluble often elude crystallization. Here, a novel strategy is presented that leads to the crystallization of the isolated N-terminal propeptide of human procathepsin S, a proteinase belonging to the cathepsin L-like endopeptidases of the clan CA1 cysteine peptidases. Being very hydrophobic, the propeptide is extremely poorly soluble in aqueous solvents at neutral pH. Solubility is much better at acidic pH, but the native structure is destroyed under these conditions. A novel approach to the crystallization of this poorly soluble protein is presented in which it is first unfolded in an acidic buffer (pH 4.5) and then mixed with a nearly neutral crystallization buffer (pH 6.75) in which the native conformation should form spontaneously. Crystals were grown at a high concentration of MES (1.14 M) with 10% 2-propanol as precipitant. They belong to a tetragonal space group, with unit-cell parameters a = b = 151.1, c = 75.8 A. Diffraction data to a resolution of 3.5 A were obtained.
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