Inhibition of trypsin and thrombin by amino(4-amidinophenyl)methanephosphonate diphenyl ester derivatives: X-ray structures and molecular models.

X-ray structures of trypsin from bovine pancreas inactivated by diphenyl [N-(benzyloxycarbonyl)amino](4-amidinophenyl)methanephosphonate [Z-(4-AmPhGly)P(OPh)2] were determined at 113 and 293 K to 1.8 angstrom resolution and refined to R factors of 0.211 (113 K) and 0. 178 (293 K). The structures reveal a tetrahedral phosphorus covalently bonded to the O gamma of the active site serine. Covalent bond formation is accompanied by the loss of both phenoxy groups. The D-stereoisomer of Z-(4-AmPhGly)P-(OPh)2 is not observed in the complex. The L-stereoisomer of the inhibitor forms contacts with several residues in the trypsin active site. One of the phosphonate oxygens is inserted into the oxyanion hole and forms hydrogen bonds to the amides of Gly193, Asp194, and Ser195. The second phosphonate oxygen forms hydrogen bonds to N epsilon 2 of His 57. The p-amidinophenylglycine moiety binds into the trypsin primary specificity pocket, interacting with Asp189. The amide forms a hydrogen bond to the carbonyl oxygen atom of Ser214. The inhibitor moiety, from the 113 K structure of trypsin inactivated by the reaction product of Z-(4-AmPhGly)P(OPh)2, was docked into human thrombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., & Hofsteenge, J. (1989) EMBO J. 8, 3467-3475] and energy minimized. The inhibitor fits well into the thrombin active site, forming favorable contacts similar to those in the trypsin complex with no bad contacts.

Mechanism-based isocoumarin inhibitors for blood coagulation serine proteases. Effect of the 7-substituent in 7-amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on inhibitory and anticoagulant potency.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.

X-ray crystal structure of a pea lectin-trimannoside complex at 2.6 A resolution.

The x-ray crystal structure of pea lectin, in complex with a methyl glycoside of the N-linked-type oligosaccharide trimannosyl core, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, has been solved by molecular replacement and refined at 2.6-A resolution. The R factor is 0.183 for all data in the 8.0 to 2.6 A resolution range with an average atomic temperature factor of 26.1 A2. Strong electron density for a single mannose residue is found in the monosaccharide-binding site suggesting that the trisaccharide binds primarily through one of the terminal alpha-linked mannose residues. The complex is stabilized by hydrogen bonds involving the protein residues Asp-81, Gly-99, Asn-125, Ala-217, and Glu-218, and the carbohydrate oxygen atoms O3, O4, O5, and O6. In addition, the carbohydrate makes van der Waals contacts with the protein, involving Phe-123 in particular. These interactions are very similar to those found in the monosaccharide complexes with concanavalin A and isolectin 1 of Lathyrus ochrus, confirming the structural relatedness of this family of proteins. Comparison of the pea lectin complex with the unliganded pea lectin and concanavalin A structures indicates differences in the conformation and water structure of the unliganded binding sites of these two proteins. Furthermore, a correlation between the position of the carbohydrate oxygen atoms in the complex and the bound water molecules in the unliganded binding sites is found. Binding of the trimannose core through a single terminal monosaccharide residue strongly argues that an additional fucose-binding site is responsible for the high affinity pea lectin-oligosaccharide interactions.

Inhibition of porcine pancreatic elastase by 7-substituted 4-chloro-3-ethoxyisocoumarins: structural characteristics of modeled noncovalent complexes relate to the measured inhibition kinetics.

Kinetic measurements for the inhibition of porcine pancreatic elastase by 7-substituted 4-chloro-3-ethoxyisocoumarins were performed. To obtain possible explanations for the kinetic results, structures resulting from energy minimizations of inhibitor-enzyme complex structures where each inhibitor was initially positioned in 64 locations within the active site were obtained. In keeping with solution NMR studies, a positive-charged His-57 was employed. The number of low energy complex structures with Ser-195 O gamma-inhibitor benzoyl ester carbonyl carbon distances < or = 2.9 A, Ser-195 O gamma-inhibitor benzoyl ester carbonyl carbon-inhibitor benzoyl ester carbonyl oxygen angles > 91 degrees, and the inhibitor in the oxyanion hole exhibits a direct linear relationship to ln(Ki/k3). The proportion of those structures that show 7-substituent H-bonding between the inhibitor and porcine pancreatic elastase exhibits a direct relationship to k3. Assuming a direct linear relationship to ln(k3) and k3, finer differences in k3 than are experimentally observed are expected. The relationship with k3 and that with Ki/k3 are shown to be useful tools for the design of more potent 7-substituted 4-chloro-3-ethoxyisocoumarins. A novel inhibitor of this class (4-chloro-3-ethoxy-7-[(2-methyl-2- butylcarbamoyl)amino]isocoumarin) expected to be more potent is synthesized and tested. Its potency within experimental error is as predicted. Although the relationship observed with Ki/k3 involves only a twofold increase in Ki/k3 (a statistically significant increase), results with the novel inhibitor show the relationship to be valid over a four- to fivefold increase.

Michaelis complexes of porcine pancreatic elastase with 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins: translational sampling of inhibitor position and kinetic measurements.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.

Design, expression, and crystallization of recombinant lectin from the garden pea (Pisum sativum).

The propeptide form of the lectin from the garden pea (Pisum sativum agglutinin) has been expressed in Escherichia coli by attaching its cDNA to an inducible promoter. By a number of criteria, including the ability to form dimers, hemagglutination titer, Western blot, and enzyme-linked immunosorbent assay, the resulting propeptide molecule is virtually indistinguishable from the mature proteolytically processed lectin isolated from peas. Preliminary crystallization experiments using the recombinant propeptide lectin yield crystals in space group P2(1)2(1)2(1) with a = 64.8 A, b = 73.8 A, and c = 109.0 A (1 A = 0.1 nm) that diffract to 2.8-A resolution. This unit cell size is quite similar to the unit cell determined for native pea lectin, suggesting that the overall structure of the recombinant prolectin is virtually identical.

Preliminary crystallographic study of the alpha-D-galactose-specific lectin from jack fruit (Artocarpus integra) seeds.

Crystals of the alpha-D-galactose-specific lectin from Jack fruit (Artocarpus integra) have been obtained from polyethylene glycol 400 solutions. The crystals are orthorhombic, space group P2(1)2(1)2 with a = 77.09 A, b = 123.3 A and c = 78.73 A (1 A = 0.1 nm) and have one 39,500 Mr tetramer per asymmetric unit. The crystals diffract to at least 2.8 A on precession photographs.

The crystal structure of pea lectin at 3.0-A resolution.

The structure of pea lectin has been determined to 3.0-A resolution based on multiple isomorphous replacement phasing to 6.0-A resolution and a combination of single isomorphous replacement, anomalous scattering, and density modification to 3.0-A resolution. The pea lectin model has been optimized by restrained least squares refinement against the data between 7.0- and 3.0-A resolution. The final model at 3.0 A gives an R factor of 0.24 and a root mean square deviation from ideal bond distances of 0.02 A. The two monomers in the asymmetric unit are related by noncrystallographic 2-fold symmetry to form a dimer. Monomers were treated independently in modeling and refinement, but are found to be virtually identical at this resolution. The molecular structure of the pea lectin monomer is very similar to that of concanavalin A, the lectin from the jack bean. Similarities extend from secondary and tertiary structures to the occurrence of a cis-peptide bond and the pattern of coordination of the Ca2+ and Mn2+ ions. Differences between the two lectin structures are confined primarily to the loop regions and to the chain termini, which are different and give rise to the unusual permuted relationship between the pea lectin and concanavalin A protein sequences.

Preliminary X-ray investigation of variant-2 scorpion toxin from Centruroides sculpturatus Ewing. Evidence of a reversible transition between crystal forms.

Crystals of Variant-2 scorpion toxin have been grown using seeding techniques from 30% 2-methyl-2,4-pentanediol at pH 9.2 and T = 4 degrees C. These crystals display a temperature-dependent, reversible phase transition near room temperature. The apparent space group for the high-temperature form is P3121 or P3221 with a = 48.8(1) A and c = 43.7(1) A, and with one molecule per asymmetric unit. At lower temperature, the crystals undergo a phase transition in which the space group remains the same but with c' (approximately equal to 2c) = 86.1(1) A. In addition, the low-temperature form displays several weak, diffuse reflections that correspond to a tripling of the a axis. The high-temperature form diffracts beyond 1.8-A resolution and appears to be suitable for a complete structural study.

Structure of variant-3 scorpion neurotoxin from Centruroides sculpturatus Ewing, refined at 1.8 A resolution.

The three-dimensional structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been determined by X-ray diffraction data. The initial model for the 65-residue protein was obtained at 3 A resolution by multiple-isomorphous-replacement methods. The structure was refined at 1.8 A resolution by restrained difference-Fourier methods, and by free-atom, block-diagonal least-squares. Considering the 4900 reflections for which d = 1.8-7 A and Fo greater than 2.5 sigma (Fo), the final R-index is 0.16 for the restrained model, and 0.14 for the free-atom model. Average estimated errors in atomic co-ordinates are about 0.1 A. The refined structure includes 492 protein atoms; one molecule of 2-methyl-2,4-pentanediol, which is tightly bound in a hydrophobic pocket on the surface of the protein; and 72 additional solvent sites. The major secondary structural features are two and a half turns of alpha-helix and a three-strand stretch of antiparallel beta-sheet. The helix is connected to the middle strand of the beta-sheet by two disulfide bridges, and a third disulfide bridge is located nearby. Several loops extend out of this dense core of secondary structure. The protein displays several reverse turns and a highly contorted proline-rich, COOH-terminal segment. One of the proline residues (Pro59) assumes a cis-conformation. The structure involves 44 intramolecular hydrogen bonds. The crystallographic results suggest two major corrections in the published primary structure; one of these has been confirmed by new chemical sequence data. The protein displays a large flattened surface that contains a high concentration of hydrophobic residues, along with most of the conserved amino acids that are found in the scorpion neurotoxins.

The crystal structure of pea lectin at 6-A resolution.

The three-dimensional crystal structure of the mitogenic lectin from the green pea (Pisum sativum) has been determined at 6-A resolution by x-ray diffraction methods. Pea lectin was isolated by use of affinity chromatography and was crystallized from polyethylene glycol solutions. Crystals of pea lectin are orthorhombic, space group P212121, and diffract to at least 1.2-A resolution. The unit cell dimensions are a = 50.85(5), b = 61.23(5), and c = 137.3(2) A. The calculated mass of protein per asymmetric unit is 49,000 daltons, and the crystals are 44% solvent by volume. There are two pea lectin monomers per crystallographic asymmetric unit. Diffractometer data were collected from a native crystal and from a single site uranyl heavy atom derivative crystal. The position of the uranium atom, determined from three-dimensional Patterson maps, was refined by least squares techniques (R index - 0.46 for centric data). A three-dimensional electron density map was calculated by use of phases determined by isomorphous-replacement and anomalous-dispersion contributions. The boundaries of the pea lectin molecule are clearly visible in the map. The molecule appears to be a dimer, roughly peanut-shaped, formed by the close association of the two monomer units. In shape and size, it bears a striking resemblance to the concanavalin A dimer, in which monomers combine to form a dimer-wide contiguous antiparallel pleated sheet.

The three-dimensional structure of scorpion neurotoxins.

The crystal and molecular structure of a toxin from the scorpion Centruroides sculpturatus has been solved by standard x-ray crystallographic methods at 3 A resolution. Subsequently the 3 A model has been refined and the resolution has been extended to 1.8 A using the gradient-curvature method. The final reliability index of 0.17 The structure has two and a half turns of alpha-helix, a three-strand stretch of antiparallel beta-sheet and several beta-turns. Three of the four disulfide bridges are found in close interaction with the alpha-helix and beta-sheet structures in what constitutes a very rigid part of the molecule. Examination of available scorpion toxin sequences reveals several sections containing invariant and/or semiinvariant amino acids. Many of these residues are found clustered on a rather large flat surface which is also clearly more hydrophobic than other areas on the molecule. These observations suggest that this surface may play a role in the biological action of scorpion toxins. Secondary structure predictions calculated using the method of Dufton and Hider agree well with the x-ray structure. This is also true for other scorpion toxins and reinforces the idea that scorpion toxins are a family of structurally related proteins.

Three-dimensional structure of a protein from scorpion venom: a new structural class of neurotoxins.

The three-dimensional crystal structure of variant-3 toxin from the scorpion Centruroides sculpturatus Ewing has been determined at 3 A resolution. Phases were obtained by use of K2PtCl4 and K2IrCl6 derivatives. The most prominent secondary structural features are two and a half turns of alpha-helix and a three-strand stretch of antiparallel beta-sheet, which runs parallel to the alpha-helix. The helix is connected to the middle strand of the beta-sheet by two disulfide bridges; a third disulfide bridge is located nearby. Several loops extend out of this dense core of secondary structure. The largest loop is joined to the COOH terminus of the molecule by the fourth disulfide bridge. The overall shape of the molecule resembles a right-hand fist: the alpha-helix runs along the knuckles of the fist; the beta-sheet lies along the second and third joints of the fingers; the thumb is defined by two short loops that are composed of residues 16-21 and residues 41-46; the wrist corresponds to the COOH-terminal stretch of residues 52-65 and a loop composed of residues 5-14; and the second joint of the little finger is near the NH2 terminus of the molecule. The alpha-carbon backbone displays a large flat surface that lies along the second joints of the fingers and the heel of the hand in the fist model. Several of the conserved residues in the scorpion neurotoxins are clustered on this surface, which may play a role in interactions of scorpion toxins with sodium channels of excitable membranes.

Yeast phenylalanine transfer RNA: atomic coordinates and torsion angles.

The atomic coordinates of yeast phenylalanine transfer RNA (tRNA) as well as the torsion angles of the polynucleotide chain are presented as derived from an x-ray diffraction analysis of orthorhombic crystals. A comparison is made between the coordinates obtained from analysis of monoclinic crystals of the same material. It is concluded that the molecule has substantially the same form in the orthorhombic and the monoclinic lattices, except for differences found between residues at the 3' end of the polynucleotides chain. A number of observations are made concerning hydrogen bonding interactions which may account for many of the residues conserved in all tRNA sequences.