Molecular structure of the α-lytic protease from Myxobacter 495 at 2·8 Å resolution☆

Abstract The α-lytic protease was isolated from an extracellular filtrate of the soil microorganism Myxobacter 495. Trigonal crystals (space group, P 3 2 21) of this serine enzyme were grown from 1·3 m -Li 2 SO 4 at pH 7·2. X-ray reflections from crystals of the native enzyme, comprising the 2·8 A limiting sphere, were phased by the multiple isomorphous replacement technique. Five heavy-atom derivatives were used and the overall mean figure of merit 〈 m 〉 is 0·83. The resulting native electron density map of α-lytic protease has been interpreted in conjunction with the published sequence (Olson et al. , 1970) of 198 amino-acid residues. α-Lytic protease has a structural core similar to that of the pancreatic serine proteases (108 α-carbon atom positions are topologically equivalent (within 2·0 A) to residues of porcine elastase) and its tertiary structure is even more closely related to the two other bacterial serine protease structures previously determined (James et al. , 1978; Brayer et al. , 1978b; Delbaere et al. , 1979a). α-Lytic protease has the following distinctive features in common with the bacterial serine enzymes, Streptomyces griseus proteases A and B: an amino terminus that is exposed to solvent on the enzyme surface, a considerably shortened uranyl loop (residues 65 to 84), a major segment of polypeptide chain from the autolysis loop deleted (residues 144 to 155), a buried guanidinium group of Arg138 in an ion-pair bond with Asp194, and an altered conformation of the methionine loop (residues 168 to 182) relative to the pancreatic enzymes. At the present resolution, the members of the catalytic quartet (Ser214, Asp102, His57 and Ser195) adopt the conformation found in all members of the Gly-Asp-Ser-Gly-Gly serine protease family. The carboxylate of Asp102 is in a highly polar environment, as it is the recipient of four hydrogen bonds. The interaction between the N e 2 atom of the imidazole ring in His57 and O γ atom of Ser195 is very weak (3·3 A) and supports the concept that there is little, if any, enhanced nucleophilicity of the side-chain of Ser195 in the native enzyme. The molecular basis for the observed substrate specificity of α-lytic protease is clear from the distribution of amino acid side-chains in the neighborhood of the active site. An insertion of five residues at position 217, and the conformation of the side-chain of Met192 account for the fact that the specificity pocket can bind only small residues, such as Ala, Ser or Val.