A wild-type human procathepsin B was expressed, crystallized in two crystal° forms and its crystal structure determined at 3.2 and 3.3 A resolution. The structure reveals that the propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. The structure of the enzymaticaily active domains is virtually identical to that of the native enzyme [Musil et al. (1991) EMBO J. 10, 2321-2330]: the main difference is that the occluding loop residues are lifted above the body of the mature enzyme, supporting the propeptide structure.

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Cathepsin S is unique among mammalian cysteine cathepsins in being active and stable at neutral pH. We show that autocatalytic activation of procathepsin S at low pH is a bimolecular process that is considerably accelerated (~20-fold) by glycosaminoglycans and polysaccharides such as dextran sulfate, chondroitin sulfates A and E, and dermatan sulfate through electrostatic interaction with the proenzyme. Procathepsin S is also shown to undergo autoactivation at neutral pH in the presence of dextran sulfate with t 1/2 of $20 min at pH 7.5. This novel property of procathepsin S may have implications in pathological conditions associated with the appearance of active cathepsins outside lysosomes.

A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/1 E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three‐amino‐acid extension at its N‐terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.

SummaryCaspases are a family of cysteine-dependent proteases known to be involved in the process of programmed cell death in metazoans. Recently, cyanobacteria were also found to contain caspase-like proteins, but their existence has only been identified in silico up to now. Here, we present the first experimental characterisation of a prokaryotic caspase homologue. We have expressed the putative caspase-like gene MaOC1 from the toxic bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli. Kinetic characterisation showed that MaOC1 is an endopeptidase with a preference for arginine in the P1 position and a pH optimum of 7.5. MaOC1 exhibited high catalytic rates with the k cat/KM value for Z-RR-AMC substrate of the order 10 6 M −1 s −1. In contrast to plant or metazoan caspase-like proteins, whose activity is calcium-dependent or requires dimerisation for activation, MaOC1 was activated by autocatalytic processing after residue Arg219, which separated the catalytic domain and the remaining 55 kDa subunit. The Arg219Ala mutant was resistant to autoprocessing and exhibited no proteolytic activity, confirming that processing of MaOC1 is a prerequisite for its activity. Due to their structural and functional differences to other known caspase-like proteins, we suggest to name these evolutionary primitive proteins orthocaspases.

Summary Cathepsin B and other cysteine proteases are synthesized as zymogens, which are processed to their mature forms autocatalytically or by other proteases. Autocatalytic processing was suggested to be a bimolecular process, whereas initiation of the processing has not been clarified as yet. Procathepsin B was shown by zymography to hydrolyze the synthetic substrate Z-Arg-Arg-AMC, suggesting that procathepsin B is catalytically active. The activity based probe DCG-04, which is an E-64 type inhibitor, was found to label both mature cathepsin B and its zymogen, confirming zymography data. Mutation analyses in the linker region between the propeptide and the mature part revealed that autocatalytic processing of procathepsin B is largely unaffected by mutations in this region, including mutations to prolines. Based on these results a model for autocatalytic activation of cysteine cathepsins is suggested, involving propeptide dissociation from the active-site cleft as the first step during zymogen activation. This unimolecular conformational change is followed by a bimolecular proteolytic removal of the propeptide, which can be accomplished in one or more steps. Such activation, which can be also facilitated by glycosaminoglycans or by binding to negatively charged surfaces, may have important physiological consequences, as cathepsin zymogens were often found secreted in various pathological states.

Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.