A GLUTAMIC-ACID SPECIFIC SERINE-PROTEASE UTILIZES A NOVEL HISTIDINE TRIAD IN SUBSTRATE-BINDING

Proteases specific for cleavage after acidic residues have been implicated in several disease states, including epidermolysis, inflammation, and viral processing. A serine protease with specificity toward glutamic acid substrates (Glu-SGP) has been crystallized in the presence of a tetrapeptide ligand and its structure determined and refined to an R-factor of 17% at 2.0-angstrom resolution. This structure provides an initial description of the design of proteolytic specificity for negatively charged residues. While the overall fold of Glu-SGP closely resembles that observed in the pancreatic-type serine proteases, stabilization of the negatively charged substrate when bound to this protein appears to involve a more extensive part of the protease than previously observed. The substrate carboxylate is bound to a histidine side chain, His213, which provides the primary electrostatic compensation of the negative charge on the substrate, and to two serine hydroxyls, Ser192 and Ser216. Glu-SGP displays maximum activity at pH 8.3, and assuming normal pK(a)'s, the glutamate side chain and His213 will be negatively charged and neutral, respectively, at this pH. In order for His213 to carry a positive charge at the optimal pH, its pK(a) will have to be raised by at least two units. An alternative mechanism for substrate charge compensation is suggested that involves a novel histidine triad, His213, His199, and His228, not observed in any other serine protease. The C-terminal alpha-helix, ubiquitous to all pancreatic-type proteases, is directly linked to this histidine triad and may also play a role in substrate stabilization. The amino acid sequence of this protease near the primary substrate binding site has been compared with those of distantly related viral and bacterial proteases. These considerations and comparison with a recently reported structure of a chymotrypsin-like protease from Sindbis virus core protein (Tong et al., 1993) tentatively suggest that a putative general substrate binding scheme for proteases with specificity toward glutamic acid may involve a histidine residue, His213, and a hydroxyl function at residue 192.