The high‐resolution X‐ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech <i>Hirudo medicinalis</i>

Triclinic crystals of the complex formed by eglin with subtilisin Carlsberg were analyzed by X-ray diffraction. The crystal and molecular structure of this complex was determined with data that extended to 0.12-nm resolution by a combination of Patterson search methods and isomorphous replacement techniques. Its structure was refined to a crystallographic R value of 0.178 (1.0-0.12 nm) using an energy-restraint least-squares procedure. The complete subtilisin molecule could be traced without ambiguity in the refined electron density. The eglin component, from which an amino-terminal segment is cleaved off, is only defined from Lys8I (i.e. the lysine residue 8 of the inhibitor) onwards. Per unit cell, 436 fixed solvent molecules and 2 calcium ions were located. In spite of 84 amino acid replacements and one deletion, subtilisin Carlsberg exhibits a very similar polypeptide fold to subtilisin BPN'. The root-mean-square deviations of all alpha-carbon atoms (excluding those at the deletion site) from models of subtilisin BPN' [Alden, R. A., Birktoft, J. J., Kraut, J., Robertus, J. D. & Wright, C. S. (1971) Biochem. Biophys. Res. Commun. 45, 337-344] and subtilisin Novo [Drenth, J., Hol, W. G. J., Jansonius, J. N. & Kockoek, R. (1972) Eur. J. Biochem. 25, 177-181] are 0.077 nm and 0.103 nm. Most of these deviations result from global shifts rather than changes of the local geometry. The single-residue deletion at position 56 affects only the surrounding conformation. Two sites of high electron density and close distances to surrounding oxygen ligands have been found in the Carlsberg enzyme which are probably occupied by calcium ions. Eglin consists of a twisted four-stranded beta-sheet flanked by an alpha-helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I-Asp46I, this loop is mainly stabilized by electrostatic/hydrogen bond interactions with the side chains of two arginine residues which project from the hydrophobic core [Bode, W., Papamokos, E., Musil, D., Seemüller, W. & Fritz, H. (1986) EMBO J. 5, 813-818]. The reactive site loop conformation resembles that found in other 'small' proteinase inhibitors. The scissile peptide bond is not cleaved but its carbonyl group is slightly distorted from planar geometry. Most of the intermolecular contacts are contributed by the nine residues of the reactive-site loop Gly40I-Arg48I.(ABSTRACT TRUNCATED AT 400 WORDS)