Crystal and molecular structure of the bovine α-chymotrypsin-eglin c complex at 2.0 Å resolution☆

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.

[1]  K. Merz,et al.  Theoretical examination of the mechanism of aldose-ketose isomerization. , 1993, Protein engineering.

[2]  M. James,et al.  Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-1 from Russet Burbank potato tubers at 2.1 A resolution. , 1989, Journal of molecular biology.

[3]  M. Eulitz,et al.  Structure of the elastase-cathepsin G inhibitor of the leech Hirudo medicinalis. , 1980, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[4]  J. Sussman,et al.  Gamma-chymotrypsin is a complex of alpha-chymotrypsin with its own autolysis products. , 1991, Biochemistry.

[5]  C. Betzel,et al.  Molecular dynamics refinement of a thermitase-eglin-c complex at 1.98 A resolution and comparison of two crystal forms that differ in calcium content. , 1989, Journal of molecular biology.

[6]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[7]  T. A. Jones,et al.  A graphics model building and refinement system for macromolecules , 1978 .

[8]  P. Sieber,et al.  A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudo medicinalis and its expression in E. coli. , 1984, Nucleic acids research.

[9]  G. N. Ramachandran,et al.  Stereochemical criteria for polypeptide and protein chain conformations. II. Allowed conformations for a pair of peptide units. , 1965, Biophysical journal.

[10]  M Bolognesi,et al.  Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study. , 1988, Journal of enzyme inhibition.

[11]  C. Chothia,et al.  The structure of protein-protein recognition sites. , 1990, The Journal of biological chemistry.

[12]  Randy J. Read,et al.  Crystal and molecular structures of the complex of α-chymotrypsin with its inhibitor Turkey ovomucoid third domain at 1.8 Å resolution , 1987 .

[13]  J. Greer.  Comparative modeling methods: Application to the family of the mammalian serine proteases , 1990, Proteins.

[14]  J. Hejgaard,et al.  Amino acid sequence of serine protease inhibitor CI-1 from barley. Homology with barley inhibitor CI-2, potato inhibitor I, and leech eglin , 1982 .

[15]  Djordje Musil,et al.  The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis Structural analysis, subtilisin structure and interface geometry , 1987 .

[16]  M. Laskowski,et al.  Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor interactions with serine proteinases. , 1982, Biochemistry.

[17]  J. Chang,et al.  Elastase-cathepsin G inhibitors eglin b and eglin c differ by a single Tyr----His substitution. A micro-method for the identification of amino-acid substitution. , 1985, Biological chemistry Hoppe-Seyler.

[18]  I. Mandl.  [92] Pancreatic elastase , 1962 .

[19]  K. Ohlsson,et al.  Isolation and characterisation of a low molecular weight inhibitor (of chymotrypsin and human granulocytic elastase and cathepsin G) from leeches. , 1977, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[20]  M. James,et al.  Crystal and molecular structure of chymotrypsin inhibitor 2 from barley seeds in complex with subtilisin Novo. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Huber,et al.  Structural basis of the activation and action of trypsin , 1978 .

[22]  M. Bolognesi,et al.  Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human α-, β- and γ-thrombin; a kinetic and thermodynamic study , 1988 .

[23]  J. Thornton,et al.  Beta-turns and their distortions: a proposed new nomenclature. , 1990, Protein engineering.

[24]  J Deisenhofer,et al.  Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution. , 1974, Journal of molecular biology.

[25]  M. Bolognesi,et al.  Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects". , 1983, The Journal of biological chemistry.

[26]  R. Huber,et al.  The Geometry of the Reactive Site and of the Peptide Groups in Trypsin, Trypsinogen and its Complexes with Inhibitors , 1983 .

[27]  V. Luzzati,et al.  Traitement statistique des erreurs dans la determination des structures cristallines , 1952 .

[28]  G. C. Fox,et al.  An alternative method of solving the layer scaling equations of Hamilton, Rollett and Sparks , 1966 .

[29]  W. Bode,et al.  Crystal structure of the thrombin‐hirudin complex: a novel mode of serine protease inhibition. , 1990, The EMBO journal.

[30]  P. Fitzgerald.  MERLOT, an integrated package of computer programs for the determination of crystal structures by molecular replacement , 1988 .

[31]  J. Sussman,et al.  γ-chymotrypsin is a complex of α-chymotrypsin with its own autolysis products , 1991 .

[32]  M. Bolognesi,et al.  Interaction between Serine (Pro)Enzymes and Macromolecular Inhibitors. Thermodynamic and Kinetic Aspects , 1987 .

[33]  R. Huber,et al.  Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution. , 1985, Journal of molecular biology.

[34]  A. Tulinsky,et al.  Comparison of the independent solvent structures of dimeric alpha-chymotrypsin with themselves and with gamma-chymotrypsin. , 1985, The Journal of biological chemistry.

[35]  M. Bolognesi,et al.  X‐ray crystal structure of the bovine α‐chymotrypsin/eglin c complex at 2.6 Å resolution , 1990, Journal of Molecular Recognition.

[36]  Brian W. Matthews,et al.  An efficient general-purpose least-squares refinement program for macromolecular structures , 1987 .

[37]  R. Huber,et al.  The structure of a complex of recombinant hirudin and human alpha-thrombin. , 1990, Science.

[38]  M. James,et al.  Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin Novo. , 1988, Biochemistry.

[39]  W. Bode,et al.  Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin. , 1986, The EMBO journal.

[40]  H. D. Flack,et al.  Absorption-weighted mean path lengths for spheres , 1978 .

[41]  P Gros,et al.  Crystallographic refinement by incorporation of molecular dynamics: thermostable serine protease thermitase complexed with eglin c. , 1989, Acta crystallographica. Section B, Structural science.

[42]  A. Tulinsky,et al.  The refinement and the structure of the dimer of alpha-chymotrypsin at 1.67-A resolution. , 1985, The Journal of biological chemistry.

[43]  G. Cohen,et al.  Refined crystal structure of gamma-chymotrypsin at 1.9 A resolution. Comparison with other pancreatic serine proteases. , 1981, Journal of molecular biology.

[44]  J. Pflugrath,et al.  Crystal orientation and X-ray pattern prediction routines for area-detector diffractometer systems in macromolecular crystallography , 1987 .

[45]  M. James,et al.  Crystal and molecular structure of the serine proteinase inhibitor CI-2 from barley seeds. , 1988, Biochemistry.

[46]  T. Creighton,et al.  Functional evolutionary divergence of proteolytic enzymes and their inhibitors. , 1989, Trends in biochemical sciences.

[47]  M. Bolognesi,et al.  Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study. , 1991, Journal of enzyme inhibition.

[48]  M. James,et al.  Crystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg , 1985, FEBS letters.

[49]  M. Bolognesi,et al.  Binding of kunitz and eglin c inhibitors to serine proteinases: thermodynamic, kinetic and molecular aspects , 1988 .

[50]  M Bolognesi,et al.  Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 A resolution. Structure solution, crystallographic refinement and preliminary structural interpretation. , 1982, Journal of molecular biology.

[51]  A. Berger,et al.  On the size of the active site in proteases. I. Papain. , 1967, Biochemical and biophysical research communications.

[52]  R. Huber,et al.  X‐ray crystal structure of the complex of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor. , 1986, The EMBO journal.

[53]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1977, Journal of molecular biology.

[54]  B. Matthews,et al.  Is gamma-chymotrypsin a tetrapeptide acyl-enzyme adduct of alpha-chymotrypsin? , 1989, Biochemistry.

[55]  D. Blow,et al.  Structure of alpha-chymotrypsin refined at 1.68 A resolution. , 1985, Journal of molecular biology.

[56]  M. Eulitz,et al.  [60] Eglin: Elastase—Cathepsin G inhibitor from leeches , 1981 .

[57]  M. Michaelis,et al.  Distinguishing characteristics between glutamate and kainic acid binding sites in brain synaptic membranes , 1980, FEBS letters.

[58]  M. Bolognesi,et al.  Preliminary crystallographic data on the complex of bovine alpha-chymotrypsin with the recombinant proteinase inhibitor eglin c from Hirudo medicinalis. , 1989, Journal of molecular biology.

[59]  I. Kato,et al.  Protein inhibitors of proteinases. , 1980, Annual review of biochemistry.