Refined 2.5 A X-ray crystal structure of the complex formed by porcine kallikrein A and the bovine pancreatic trypsin inhibitor. Crystallization, Patterson search, structure determination, refinement, structure and comparison with its components and with the bovine trypsin-pancreatic trypsin inhibit

Abstract The complex formed by porcine pancreatic kallikrein A with the bovine pancreatic trypsin inhibitor (PTI) has been crystallized at pH 4 in tetragonal crystals of space group P 4 1 2 1 2 with one molecule per asymmetric unit. Its crystal structure has been solved applying Patterson search methods and using a model derived from the bovine trypsin-PTI complex (Huber et al. , 1974) and the structure of porcine pancreatic kallikrein A (Bode et al. , 1983). The kallikrein-PTI model has been crystallographically refined to an R -value of 0·23 including X-ray data to 2·5 A. The root-mean-square deviation, including all main-chain atoms, is 0·45 A and 0·65 A for the PTI and for the kallikrein component, respectively, compared with the refined models of the free components. The largest differences are observed in external loops of the kallikrein molecule surrounding the binding site, particularly in the C-terminal part of the intermediate helix around His172. Overall, PTI binding to kallikrein is similar to that of the trypsin complex. In particular, the conformation of the groups at the active site is identical within experimental error (in spite of the different pH values of the two structures). Ser195 OG is about 2·5 A away from the susceptible inhibitor bond Lys15 C and forms an optimal 2·5 A hydrogen bond with His57 NE. The PTI residues Thr11 to Ile18 and Val34 to Arg39 are in direct contact with kallikrein residues and form nine intermolecular hydrogen bonds. The reactive site Lys15 protrudes into the specificity pocket of kallikrein as in the trypsin complex, but its distal ammonium group is positioned differently to accommodate the side-chain of Ser226. Ser226 OG mediates the ionic interaction between the ammonium group and the carboxylate group of Asp189. Model-building studies indicate that an arginine side-chain could be accommodated in this pocket. The PTI disulfide bridge 14–38 forces the kallikrein residue Tyr99 to swing out of its normal position. Model-building experiments show that large hydrophobic residues such as phenylalanine can be accommodated at this (S2) site in a wedge-shaped hydrophobic cavity, which is formed by the indole ring of Trp215 and by the phenolic side-chain of Tyr99, and which opens towards the bound inhibitor/substrate chain. Arg17 in PTI forms a favorable hydrogen bond and van der Waals' contacts with kallikrein residues, whereas the additional hydrogen bond formed in the trypsin-PTI complex between Tvr39 OEH and Ile19 N is not possible The kallikrein binding site offers a qualitative explanation of the unusual binding and cleavage at the N-terminal Met-Lys site of kininogen. Model-building experiments suggest that the generally restricted capacity of kallikrein to bind protein inhibitors with more extended binding segments might be explained by steric hindrance with some extruding external loops surrounding the kallikrein binding site (Bode et al. , 1983).

[1]  J. Markley.  Hydrogen bonds in serine proteinases and their complexes with protein proteinase inhibitors. Proton nuclear magnetic resonance studies. , 1978, Biochemistry.

[2]  R. Geiger,et al.  Progressive inhibition of human glandular (urinary) kallikrein by human serum and identification of the progressive antikallikrein as alpha 1-antitrypsin (alpha 1-protease inhibitor). , 1981, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[3]  R. Stroud,et al.  Difference Fourier refinement of the structure of DIP‐trypsin at 1.5 Å with a minicomputer technique , 1977 .

[4]  H. Kraut,et al.  Der Nachweis eines Kreislaufhormons in der Pankreasdrüse. (IV. Mitteilung über dieses Kreislaufhormon.) , 1930 .

[5]  E. Vahtera,et al.  Absence of binding of pancreatic and urinary kallikreins to alpha 2-macroglobulin. , 1976, Biochemical Journal.

[6]  R. Huber,et al.  Crystallization, crystal structure analysis and molecular model of the third domain of Japanese quail ovomucoid, a Kazal type inhibitor. , 1981, Journal of molecular biology.

[7]  M. L. Bender,et al.  MECHANISM OF ACTION OF PROTEOLYTIC ENZYMES. , 1965, Annual review of biochemistry.

[8]  R. A. Crowther,et al.  A method of positioning a known molecule in an unknown crystal structure , 1967 .

[9]  George M. Church,et al.  A structure-factor least-squares refinement procedure for macromolecular structures using constrained and restrained parameters , 1977 .

[10]  J. Engel,et al.  The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence. , 1975, European journal of biochemistry.

[11]  C. Kutzbach,et al.  Kallikrein from pig pancreas. Purification, separation of components A and B, and crystallization. , 1972, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[12]  T. Suzuki,et al.  Bovine plasma high molecular weight kininogen: The amino acid sequence of fragment 1 (glycopeptide) released by the action of plasma kallikrein and its location in the precursor protein , 1976, FEBS letters.

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

[14]  W. Bode,et al.  The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. II. The binding of the pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially related peptides to trypsinogen and to p-guanidinobenzoate-trypsinogen. , 1979, Journal of molecular biology.

[15]  G. Cohen,et al.  Substrate binding site in bovine chymotrypsin A-gamma. A crystallographic study using peptide chloromethyl ketones as site-specific inhibitors. , 1971, Biochemistry.

[16]  G. A. Sim,et al.  The distribution of phase angles for structures containing heavy atoms. II. A modification of the normal heavy‐atom method for non‐centrosymmetrical structures , 1959 .

[17]  F. Fiedler,et al.  Inhibition of three porcine glandular kallikreins by chloromethyl ketones. , 1977, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[18]  E. Shaw,et al.  Active site mapping of human and rat urinary kallikreins by peptidyl chloromethyl ketones. , 1980, Archives of biochemistry and biophysics.

[19]  C. Chothia.  Structural invariants in protein folding , 1975, Nature.

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

[21]  D. Blow,et al.  The detection of sub‐units within the crystallographic asymmetric unit , 1962 .

[22]  C Chothia,et al.  Stability and specificity of protein-protein interactions: the case of the trypsin-trypsin inhibitor complexes. , 1976, Journal of molecular biology.

[23]  M. Rocha e Silva,et al.  Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. , 1949, The American journal of physiology.

[24]  W. Bode,et al.  The refined crystal structure of bovine beta-trypsin at 1.8 A resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7.0. , 1975, Journal of molecular biology.

[25]  L. Juliano,et al.  The cleavage of the Met-Lys bond in a bradykinin derivative by glandular kallikreins. , 1981, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[26]  H. Fritz,et al.  Zur Identität des Progressiv-Antikallikreins mit α-Antitrypsin aus Humanserum , 1969 .

[27]  A Merli,et al.  Reactivity of ferric Aplysia and sperm whale myoglobins towards imidazole. X-ray and binding study. , 1982, Journal of molecular biology.

[28]  Thomas A. Steitz,et al.  Structure of crystalline α-chymotrypsin: III. Crystallographic studies of substrates and inhibitors bound to the active site of α-chymotrypsin , 1969 .

[29]  R. Huber,et al.  Crystallographic refinement of Japanese quail ovomucoid, a Kazal-type inhibitor, and model building studies of complexes with serine proteases. , 1982, Journal of molecular biology.

[30]  C. Chothia,et al.  Principles of protein–protein recognition , 1975, Nature.

[31]  W. Bode,et al.  Refined 2 A X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin. , 1983, Journal of molecular biology.

[32]  F. Lottspeich,et al.  Human urinary kallikrein—Biochemistry and physiological studies , 1980 .

[33]  L. Delbaere,et al.  Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B. , 1980, Journal of molecular biology.

[34]  H. Jering,et al.  Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties. , 1976, European journal of biochemistry.

[35]  P. Schwager,et al.  A simple empirical absorption‐correction method for X‐ray intensity data films , 1973 .

[36]  P. Schwager,et al.  Refinement of setting angles in screenless film methods , 1975 .

[37]  H. Tschesche,et al.  The primary structure of porcine glandular kallikreins. , 1979, Advances in experimental medicine and biology.

[38]  R. Huber,et al.  The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution. , 1978, Journal of molecular biology.

[39]  W. Bode,et al.  The refined crystal structure of bovine β-trypsin at 1·8 Å resolution , 1975 .

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

[41]  I. C. O. B. Nomenclature.  IUPAC-IUB Commission on Biochemical Nomenclature. Abbreviations and symbols for the description of the conformation of polypeptide chains. Tentative rules (1969). , 1970, Biochemistry.

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

[43]  B. Lee,et al.  The interpretation of protein structures: estimation of static accessibility. , 1971, Journal of molecular biology.

[44]  Michael Levitt,et al.  Refinement of Large Structures by Simultaneous Minimization of Energy and R Factor , 1978 .

[45]  R. Geiger,et al.  Inhibition of porcine glandular kallikreins by structurally homologous proteinase inhibitors of the Kunitz (Trasylol) type. Significance of the basic nature of amino acid residues in subside positions for kallikrein inhibition. , 1979, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[46]  R M Sweet,et al.  Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6-A resolution. , 1974, Biochemistry.

[47]  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.

[48]  J. Deisenhofer,et al.  Crystallographic refinement of the structure of bovine pancreatic trypsin inhibitor at l.5 Å resolution , 1975 .

[49]  F. Fiedler,et al.  Substrate specificity of porcine pancreatic kallikrein. , 1979, Advances in experimental medicine and biology.

[50]  S. Hirono,et al.  Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution. , 1979, Journal of molecular biology.

[51]  E. Lattman,et al.  Representation of phase probability distributions for simplified combination of independent phase information , 1970 .