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'. Eglin consists of a twisted four-stranded p-sheet flanked by an a-helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I-Asp461, 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., M u d , D., Seemiiller, W. & Fritz, H. (1986) EMBO J. 5, 813 -8181. The reactivesite 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. The hydrophobic side chains of Pro421 (P4), Leu451 (Pl) and Leu471 (P2'), which are exposed to solvent in the free inhibitor, are only partially buried in the complex and are still in van der Waals contact with several solvent molecules.
Correspondence to W. Bode, Max-Planck-Institut fur Biochemie, D-8033 Martinsried bei Munchen, Federal Republic of GermanyNote. This is the second paper of a series; the first paper appeared elsewhere [33].Abbreviations. rms, root-mean-square; I, structure factor intensity; I Fobs 1, observed structure factor amplitude; IFcalc 1, calculated structure factor amplitude; PTI, pancreatic trypsin inhibitor; HLE, human leukocyte elastase; SGPB, Streptomyces griseus proteinase B; OMTKY3, third domain of the turkey ovomucoid inhibitor; OMSVP3, third domain of the silver pheasant ovomucoid inhibitor; S S I , Streptomyces subtilisin inhibitor; (21-2, chymotrypsin inhibitor 2 from barley seeds.Nomenclature. The peptide and subsite nomenclature is that suggested by Schechter and Berger [l]: amino acid residues of substrates are numbered P1, P2, P3 etc. towards the amino terminus, and PI', P2', P3' etc. towards the carboxy terminus from the reactive-site bond; the complementary subsites of the enzyme are numbered S1, S2, S3 etc. and Sl', S2', S3' etc., respectively. Inhibitor residues are designated by an I after the sequence number.Subtilisins form a group of serine...