Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 A resolution. Structural basis of Janus-faced serine protease inhibitor specificity

Understanding molecular recognition on a structural basis is an objective with broad academic and applied significance. In the complexes of serine proteases and their proteinaceous inhibitors, recognition is governed mainly by residue P1 in accord with primary serine protease specificity. The bifunctional soybean Bowman-Birk inhibitor (sBBI) should, therefore, interact at LysI16 (subdomain 1) with trypsin and at LeuI43 (subdomain 2) with chymotrypsin. In contrast with this prediction, a 2:1 assembly with trypsin was observed in solution and in the crystal structure of sBBI in complex with trypsin, determined at 2.3 A resolution by molecular replacement. Strikingly, P1LeuI43 of sBBI was fully embedded into the S(1) pocket of trypsin in contrast to primary specificity. The triple-stranded beta-hairpin unique to the BBI-family and the surface loops surrounding the active site of the enzyme formed a protein-protein-interface far extended beyond the primary contact region. Polar residues, hydrophilic bridges and weak hydrophobic contacts were predominant in subdomain 1, interacting specifically with trypsin. However, close hydrophobic contacts across the interface were characteristic of subdomain 2 reacting with both trypsin and chymotrypsin. A Met27Ile replacement shifted the ratio with trypsin to the predicted 1:1 ratio. Thus, the buried salt-bridge responsible for trypsin specificity was stabilised in a polar, and destabilized in a hydrophobic, environment. This may be used for adjusting the specificity of protease inhibitors for applications such as insecticides and cancer chemopreventive agents.     

The structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor

The structure of the complex between anhydro-trypsin and pancreatic trypsin inhibitor has been determined by difference Fourier techniques using phases obtained from the native complex (Huber et al., 1974). It was refined independently by constrained crystallographic refinement at 1.9 å resolution. The anhydro-complex has Ser 195 converted to dehydro-alanine. There were no other significant structural changes. In particular, the high degree of pyramidalization of the C atom of Lys 15 (I) of the inhibitor component observed in the native complex is maintained in the anhydro-species.     

X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.     

Crystal structure of Escherichia coli UDPMurNAc-tripeptide d-alanyl-d-alanine-adding enzyme (MurF) at 2.3 A resolution

MurF is required to catalyze the final step in the synthesis of the cytoplasmic precursor of the bacterial cell wall peptidoglycan, rendering it an attractive target for antibacterial drug development. The crystal structure of the MurF apo-enzyme has been determined using the multiwavelength anomalous dispersion method and refined to 2.3 A resolution. It contains three consecutive open alpha/beta-sheet domains. In comparison with the complex crystal structures of MurD and its substrates, The topology of the N-terminal domain of MurF is unique, while its central and C-terminal domains exhibit similar mononucleotide and dinucleotide-binding folds, respectively. The apo-enzyme of MurF crystal structure reveals an open conformation with the three domains juxtaposed in a crescent-like arrangement creating a wide-open space where substrates are expected to bind. As such, catalysis is not feasible and significant domain closure is expected upon substrate binding.     

Crystal structure of a barnase-d(GpC) complex at 1.9 A resolution

The ribonuclease excreted by Bacillus amyloliquefaciens, Barnase, was co-crystallized with the deoxy-dinucleotide d(GpC). The crystal structure was determined by molecular replacement from a model of free Barnase previously derived by Mauguen et al. Refinement was carried out using data to 1.9 A resolution. The final model, which has a crystallographic R factor of 22%, includes 869 protein atoms, 38 atoms from d(GpC), a sulfate ion and 73 water molecules. Only minor differences from free Barnase are seen in the protein moiety, the root-mean-square C alpha movement being 0.45 A. The dinucleotide has a folded conformation. It is located near the active site of the enzyme, but outside the protein molecule and making crystal packing contacts with neighboring molecules. The guanine base is stacked on the imidazole ring of active site His102, rather than binding to the so-called recognition loop as it does in other complexes of guanine nucleotides with microbial nucleases. The deoxyguanosine is syn, with the sugar ring in C-2'-endo conformation; the deoxycytidine is anti and C-4'-exo. In addition to the stacking interaction, His102 hydrogen bonds to the free 5' hydroxyl, which is located near the position where the 3' phosphate group is found in other inhibitors of microbial ribonucleases. While the mode of binding observed with d(GpC) and Barnase would be non-productive for a dinucleotide substrate, it may define a site for the nucleotide product on the 3' side of the hydrolyzed bond.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region

The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 Å resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution

The crystal structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor has been refined with data to 1.9 Å resolution, using a procedure described by Deisenhofer &; Steigemann (1974) in their refinement of the crystal structure of the free inhibitor. This procedure involves cycles consisting of phase calculation using the current atomic model, Fourier synthesis using these phases and the observed structure factor amplitudes and Diamond's real-space refinement (Diamond, 1971,1974). At various stages, difference Fourier syntheses are calculated to detect and correct gross errors in the model and to localize solvent molecules.The refinement progressed smoothly, starting with the model obtained from the isomorphous Fourier map at 2.6 Å resolution. The R-factor is 0.23 for 20,500 significantly measured reflections to 1.9 Å resolution, using an over-all temperature factor of 20 Ų. The estimated standard deviation of atomic positions is 0.09 Å.An objective assessment of the upper limit of the error in the atomic coordinates of the final model is possible by comparing the inhibitor component in the model of the complex with the refined structure of the free inhibitor (Deisenhofer &; Steigemann, 1974). The mean deviation of main-chain atoms of the two molecular models in internal segments is 0.25 Å, of main-chain dihedral angles 5.1 ° and side-chain dihedral angles 6.5 °.A comparison of the trypsin component with α-chymotrypsin (Birktoft &; Blow, 1972) showed a mean deviation of main-chain atoms of 0.75 Å. The structures are closely similar and the various deletions and insertions cause local structural differences only.     

Crystal structure of human purine nucleoside phosphorylase at 2.3A resolution

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. In human, PNP is the only route for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and its low resolution structure has been used for drug design. Here we report the structure of human PNP solved to 2.3A resolution using synchrotron radiation and cryocrystallographic techniques. This structure allowed a more precise analysis of the active site, generating a more reliable model for substrate binding. The higher resolution data allowed the identification of water molecules in the active site, which suggests binding partners for potential ligands. Furthermore, the present structure may be used in the new structure-based design of PNP inhibitors.     

The three-dimensional structure of recombinant bovine chymosin at 2.3 A resolution

The crystal structure of recombinant bovine chymosin (EC 3.4.23.4; renin), which was cloned and expressed in Escherichia coli, has been determined using X-ray data extending to 2.3 A resolution. The crystals of the enzyme used in this study belong to the space group I222 with unit cell dimensions alpha = 72.7 A, b = 80.3 A, and c = 114.8 A. The structure was solved by the molecular replacement method and was refined by a restrained least-squares procedure. The crystallographic R factor is 0.165 and the deviation of bond distances from ideality is 0.020 A. The resulting model includes all 323 amino acid residues, as well as 297 water molecules. The enzyme has an irregular shape with approximate maximum dimensions of 40 x 50 x 65 A. The secondary structure consists primarily of parallel and antiparallel beta-strands with a few short alpha-helices. The enzyme can be subdivided into N- and C-terminal domains which are separated by a deep cleft containing the active aspartate residues Asp-34 and Asp-216. The amino acid residues and waters at the active site form an extensive hydrogen-bonded network which maintains the pseudo 2-fold symmetry of the entire structure. A comparison of recombinant chymosin with other acid proteinases reveals the high degree of structural similarity with other members of this family of proteins as well as the subtle differences which make chymosin unique. In particular, Tyr-77 of the flap region of chymosin does not hydrogen bond to Trp-42 but protrudes out in the P1 pocket forming hydrophobic interactions with Phe-119 and Leu-32. This may have important implications concerning the mechanism of substrate binding and substrate specificity.     

Crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) at 2.6 A resolution

The crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) was solved at 2·6 Å resolution. Each subunit of the dimeric inhibitor has a five-stranded antiparallel β-sheet and two short α-helices. The subunit-subunit interface formed by a stack of two β-sheets provided by the two subunits resembles the dimer-dimer interface of concanavalin A. Conformation of the reactive site around the scissible bond Met73-Val74 seems very rigid. Between bovine pancreatic trypsin inhibitor (Kunitz) and the Streptomyces inhibitor, the reactive site conformations are almost identical with each other from the P₂ to P₂′ residues, while between the soybean trypsin inhibitor (Kunitz) and the Streptomyces inhibitor they are similar from the P₂ to P₁′ residues. There are overall similarities in conformation extending from the P₃ to P₂′ residues between the Streptomyces inhibitor and a hypothetical substrate presumed (Robertus et al., 1972b) to be bound to subtilisin BPN′ in a productive binding mode. Apart from the reactive site, there seems to be no structural relationship among the Streptomyces, bovine pancreatic and soybean inhibitors, suggesting their convergent evolution from separate ancestral proteins.     

Crystal structure of mung bean inhibitor lysine active fragment complex with bovine beta-trypsin at 1.8A resolution.

The crystal structure of the complex of mung bean inhibitor lysine active fragment with bovine beta-trypsin has been determined by X-ray crystallographic analysis at a resolution of 1.8 A. Refinement of the model of the complex converged at a final R value of 0.16. From the resulting electron density map, about one-third of the residues of the inhibitor were identified and two residues, at position P4 and P2' respectively, were found to be inconsistent with the sequence reported previously. The peptide chain of the inhibitor at the trypsin active site turns back sharply at Pro23I and forms a 9-residue reactive loop, which interacts with trypsin in a similar manner to the other families of inhibitors, suggesting an important and common role of these regions in exhibiting inhibitory activity.     

Crystal structure of a complex formed between a snake venom phospholipase A(2) and a potent peptide inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 A resolution

Phospholipase A(2) is an important enzyme involved in the production of prostaglandins and their related compounds causing inflammatory disorders. Among the several peptides tested, the peptide Phe-Leu-Ser-Tyr-Lys (FLSYK) showed the highest inhibition. The dissociation constant (K(d)) for this peptide was calculated to be 3.57 +/- 0.05 x 10(-9) m. In order to further improve the degree of inhibition of phospholipase A(2), a complex between Russells viper snake venom phospholipase A(2) and a peptide inhibitor FLSYK was crystallized, and its structure was determined by crystallographic methods and refined to an R-factor of 0.205 at 1.8 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) (molecules A and B) and a peptide molecule specifically bound to molecule A only. The two molecules formed an asymmetric dimer. The dimerization caused a modification in the binding site of molecule A. The overall conformations of molecules A and B were found to be generally similar except three regions i.e. the Trp-31-containing loop (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Out of the above three, the most striking difference pertains to the conformation of Trp-31 in the two molecules. The orientation of Trp-31 in molecule A was suitable for the binding of FLSYK, while it disallowed the binding of peptide to molecule B. The structure of the complex clearly shows that the peptide is so placed in the binding site of molecule A that the side chain of its lysine residue interacted extensively with the enzyme and formed several hydrogen bonds in addition to a strong electrostatic interaction with critical Asp-49. The C-terminal carboxylic group of the peptide interacted with the catalytic residue His-48.     

Crystal structure of phosphodiesterase 4D and inhibitor complex(1)

Cyclic nucleotide phosphodiesterases (PDEs) regulate physiological processes by degrading intracellular second messengers, adenosine-3′,5′-cyclic phosphate or guanosine-3′,5′-cyclic phosphate. The first crystal structure of PDE4D catalytic domain and a bound inhibitor, zardaverine, was determined. Zardaverine binds to a highly conserved pocket that includes the catalytic metal binding site. Zardaverine fills only a portion of the active site pocket. More selective PDE4 inhibitors including rolipram, cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space for increased binding energy and selectivity. In the crystal structure, the catalytic domain of PDE4D possesses an extensive dimerization interface containing residues that are highly conserved in PDE1, 3, 4, 8 and 9. Mutations of R358D or D322R among these interface residues prohibit dimerization of the PDE4D catalytic domain in solution.     

Crystal structure of (L-Arg)-BO bovine insulin at 0.21 nm resolution

The crystal structure of (L-Arg)-B0 bovine insulin has been determined, using data to 0.21 nm and atomic parameters of 2Zn porcine insulin as a starting model, by the difference Fourier method, the restrained least square method and X-PLOR package, interspersed with careful review of the electron density, to a final R-factor of 0.182 and r.m.s. deviation of 0.002 2nm for the bond lengths and 4.3° for the bond angles. The electron densities of additional (L-Arg)-B0 residues to B-chain N-terminus of two monomers in each asymmetric unit are very dear. The crystallographic micro-environment of the N-terminus of the B-chain is different from that of rhombohedral 2-zinc insulin.     

The structure of denatured bovine pancreatic trypsin inhibitor (BPTI)

In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3-4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead-form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5-Cys14, Cys30-Cys38 and Cys51-Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.     

Crystal structure of a deacylation-defective mutant of penicillin-binding protein 5 at 2.3-A resolution

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a d-alanine carboxypeptidase, cleaving the C-terminal d-alanine residue from cell wall peptides. Like all PBPs, PBP 5 forms a covalent acyl-enzyme complex with beta-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acyl-enzyme complex (t(12) approximately 9 min). A Gly-105 --> Asp mutation in PBP 5 markedly impairs this beta-lactamase activity (deacylation), with only minor effects on acylation, and promotes accumulation of a covalent complex with peptide substrates. To gain further insight into the catalytic mechanism of PBP 5, we determined the three-dimensional structure of the G105D mutant form of soluble PBP 5 (termed sPBP 5') at 2.3 A resolution. The structure is composed of two domains, a penicillin binding domain with a striking similarity to Class A beta-lactamases (TEM-1-like) and a domain of unknown function. In addition, the penicillin-binding domain contains an active site loop spatially equivalent to the Omega loop of beta-lactamases. In beta-lactamases, the Omega loop contains two amino acids involved in catalyzing deacylation. This similarity may explain the high beta-lactamase activity of wild-type PBP 5. Because of the low rate of deacylation of the G105D mutant, visualization of peptide substrates bound to the active site may be possible.