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B F Edwards

Publications and source records attributed to B F Edwards.

At least 37 records · Page 2Linked to original sources
PubMed1995

1.6 A structure of semisynthetic ribonuclease crystallized from aqueous ethanol. Comparison with crystals from salt solutions and with ribonuclease A from aqueous alcohol solutions.

The non-covalent combination of residues 1-118 of RNase A with a synthetic 14-residue peptide containing residues 111-124 of the molecule forms a highly active semisynthetic enzyme, RNase 1-118:111-124. With this enzyme, the roles played by the six C-terminal residues in generating the catalytic efficiency and substrate specificity of RNase can be studied using chemically synthesized analogs. The structure of RNase 1-118:111-124 from 43% aqueous ethanol has been determined using molecular-replacement methods and refined to a crystallographic R-factor of 0.166 for all observed reflections in the range 7.0-1.6 A (Protein Data Bank file ISSC). The structure is compared with the 2.0 A structure of RNase A from 43% aqueous 2-methyl-2-propanol and with the 1.8 A structure of the semisynthetic enzyme obtained from crystals grown in concentrated salt solution. The structure of RNase 1-118:111-124 from aqueous ethanol is virtually identical to that of RNase A from aqueous 2-methyl-2-propanol. Half of the crystallographically bound water molecules are not coincident, however. The structure is somewhat less similar to that of RNase 1-118:111-124 from salt solutions, with a major difference being the positioning of active-site residue His119.

Journal Article↗
PubMed1995

The crystal structure of recombinant human neutrophil-activating peptide-2 (M6L) at 1.9-A resolution.

Neutrophil-activating peptide-2 (NAP-2) is a 70-residue carboxyl-terminal fragment of platelet basic protein, which is found in the alpha-granules of human platelets. NAP-2, which belongs to the CXC family of chemokines that includes interleukin-8 and platelet factor 4, binds to the interleukin-8 type II receptor and induces a rise in cytosolic calcium, chemotaxis of neutrophils, and exocytosis. Crystals of recombinant NAP-2 in which the single methionine at position 6 was replaced by leucine to facilitate expression belong to space group P1 (unit cell parameters a = 40.8, b = 43.8, and c = 44.7 A and alpha = 98.4 degrees, beta = 120.3 degrees, and gamma = 92.8 degrees), with 4 molecules of NAP-2 (Mr = 7600) in the asymmetric unit. The molecular replacement solution calculated with bovine platelet factor 4 as the starting model was refined using rigid body refinement, manual fitting in solvent-leveled electron density maps, simulated annealing, and restrained least squares to an R-factor of 0.188 for 2 sigma data between 7.0- and 1.9-A resolution. The final refined crystal structure includes 265 solvent molecules. The overall tertiary structure, which is similar to that of platelet factor 4 and interleukin-8, includes an extended amino-terminal loop, three strands of antiparallel beta-sheet arranged in a Greek key fold, and one alpha-helix at the carboxyl terminus. The Glu-Leu-Arg sequence that is critical for receptor binding is fully defined by electron density and exhibits multiple conformations.

Amino Acid Sequence↗
PubMed1994

Preliminary X-ray diffraction analysis of crystals of the PII protein from Escherichia coli.

PII protein, which carries metabolic signals regulating the transcription and activity of glutamine synthetase in nitrogen assimilation in Escherichia coli, has been crystallized in space group P2(1) with a = 47.8 A, b = 62.9 A, c = 52.8 A and beta = 100.3 degrees and space group P2(1)2(1)2(1) with a = 52.2 A. b = 64.9 A and c = 100.1 A. Both the monoclinic crystals, which diffract beyond 3.0 A, and the orthorhombic crystals, which diffract beyond 2.5 A, probably have three molecules of 12,400 Da each in the crystallographic asymmetric unit.

Bacterial Proteins↗
PubMed1994

Crystallization and preliminary X-ray diffraction analysis of the ArsC protein from the Escherichia coli arsenical resistance plasmid, R773.

Diffraction data to 3.0 A resolution were collected on crystals of ArsC protein from the conjugative resistance factor R773 which mediates arsenical resistance in the Gram negative bacterium Escherichia coli. The crystal system is tetragonal, a = 116.2 A, c = 145.0 A and the space group is either P4(1)2(1)2 or P4(3)2(1)2. The most probable range for the contents of the asymmetric unit is four to eight ArsC molecules (M(r) = 15,811).

Adenosine Triphosphatases↗
PubMed1994

Crystallization of hemoglobins II and III of the symbiont-harboring clam Lucina pectinata.

Diffraction data to 2.7 A resolution were measured on crystals of the homotetramers of components II and III of the cytoplasmic hemoglobin of the symbiont-harboring clam Lucina pectinata. Even though the crystallization conditions are different and the sequence homology of the two hemoglobins is only 63%, the crystals are isomorphous to each other and to the heterotetramer Hb II/III, implying that the residues primarily involved in the intermolecular interactions and responsible for crystal cohesion may be invariant.

Journal Article↗
PubMed1994

The occupancy of two distinct conformations by active-site histidine-119 in crystals of ribonuclease is modulated by pH.

Structures of a semisynthetic RNase have been obtained to a resolution of 2.0 A at pH values of 5.2, 6.5, 7.5, and 8.8, respectively. The principle structural transformation occurring over this pH range is the conversion of the side chain of active site residue His-119 from one conformation (chi 1 = -43 degrees to -57 degrees) at low pH to another (chi 1 = +159 degrees to +168 degrees) at higher pH values. On the basis of this observation, a model is proposed that reconciles the disparate pK values for His-119 in the enzyme-substrate complex that have been deduced from kinetic studies and from proton NMR measurements in the presence of pseudosubstrates.

Binding Sites↗
PubMed1994

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases.

The structures of two catalytically modified semisynthetic RNases obtained by replacing phenylalanine 120 with leucine and tyrosine have been determined and refined at a resolution of 2.0 A (R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.8-A structure (R = 0.204) of the fully active phenylalanine-containing enzyme (Martin PD, Doscher MS, Edwards BFP, 1987, J Biol Chem 262:15930-15938) and with the catalytically defective D121A (2.0 A, R = 0.172) and D121N (2.0 A, R = 0.186) analogs (deMel VSJ, Martin PD, Doscher MS, Edwards BFP, 1992, J Biol Chem 267:247-256). The movement away from the active site of the loop containing residues 65-72 is seen in all three catalytically defective analogs--F120L, D121A, and D121N--but not in the fully active (or hyperactive) F120Y. The insertion of the phenolic hydroxyl of Tyr 120 into a hydrogen-bonding network involving the hydroxyl group of Ser 123 and a water molecule in F120Y is the likely basis for the hyperactivity toward uridine 2',3'-cyclic phosphate previously found for this analog (Hodges RS, Merrifield RB, 1974, Int J Pept Protein Res 6:397-405) as well as the threefold increase in KM for cytidine 2',3'-cyclic phosphate found for this analog by ourselves.

Amino Acid Sequence↗
PubMed1993

Crystal structure of the complex of human alpha-thrombin and nonhydrolyzable bifunctional inhibitors, hirutonin-2 and hirutonin-6.

The crystal structure of the complexes of hirutonin-2 and hirutonin-6 with human alpha-thrombin have been solved and refined to R-factors of 0.169 (2.0 A resolution) and 0.162 (2.1 A), respectively. Hirutonins belong to a family of bifunctional inhibitors bearing a noncleavable moiety mimicking the scissile bond. Hirutonin-2 is an analog of (D)Phe-Pro-Arg-Gly-hirudin49-65; hirutonin-6 has the same N-terminal tripeptide connected to a shortened fibrinogen exosite-binding part by a short, nonpeptidyl linker. The hirutonin-6 molecule is well defined in the electron density with the exception of the C-terminal Leu-h61. The linker follows near the bottom of the canyon connecting the active site with the exosite, forms a short antiparallel beta-sheet-like arrangement with Leu40-Leu41 and makes van der Waals contacts with Glu39-Leu40-Leu41 of thrombin. In the thrombin-hirutonin-2 complex, the N- and C-terminal parts of the inhibitor are well ordered (except the C-terminal Gln-h65) while the central portion of the linker is partially disordered. The glycine analog in the P1' position of hirutonin-2 assumes a conformation similar to that of the canonical form (Bode and Huber (1992) Eur. J. Biochem. 204:433-451) and supports the identification of the S1' site as restricted by His57, Trp60D, Lys60F, and the Cys42-Cys58 disulfide bridge. The carbonyl oxygen of the P1 arginine residue is located in the oxyanion hole formed by the NH groups of Gly193 and Ser195, while the carbonyl carbon is positioned within a short distance, 2.8 A, from the O gamma of Ser195. This resembles the conformation of the substrate-like inhibitors bound to other serine proteases. The N-terminal (D)Phe-Pro-Arg fragment common to both inhibitors binds to thrombin in a fashion very similar to that of other inhibitors having this motif. The binding of the C-terminus of hirutonins to the fibrinogen-binding exosite is similar to that observed in hirudin and hirulog complexes.

Amino Acid Sequence↗
PubMed1992

Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin.

NMR experiments were carried out to study the interaction of thrombin with a synthetic peptide, ESKATNATLDPR, derived from the newly-identified platelet receptor for thrombin [Vu, T.-K. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. On the basis of the observation of the thrombin-induced line broadening and transferred NOEs, binding of the peptide was found to be located exclusively within residues LDPR of the proteolytic cleavage site LDPR/S essential for receptor activation by thrombin. Measurement of transferred NOEs and molecular modeling indicate that the side chain of the Asp(P3) residue may form a hydrogen bond with thrombin and, by doing so, it is brought near a positively-charged thrombin residue Arg(221A), thereby partially neutralizing the negative charge of an Asp residue at this site of protein substrates. The hydrophobic side chains of residues Leu(P4) and Pro(P2) reside on the same side of the peptide backbone as indicated by transferred NOEs and were found by modeling to fit into a hydrophobic cage around the thrombin active site. These results suggest that the interaction of thrombin with protein substrates such as prothrombin, protein C, protein S, the platelet receptor, and the A alpha- and B beta-chains of fibrinogen all follow the same canonical binding mode in that the substrate forms an antiparallel beta-strand with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.

Amino Acid Sequence↗
PubMed1992

Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics.

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.

Amidines↗
PubMed1992

The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active site of thrombin; the remaining 4 residues, which have been cleaved from the tetradecapeptide at the Arg-16f/Gly-17f bond, are not seen. Residues 7f-11f at the NH2 terminus of the peptide form a single turn of alpha-helix that is connected by Gly-12f, which has a positive phi angle, to an extended chain containing residues 13f-16f. The major specific interactions between the peptide and thrombin are 1) a hydrophobic cage formed by residues Tyr-60A, Trp-60D, Leu-99, Ile-174, Trp-215, Leu-9f, Gly-13f, and Val-15f that surrounds Phe-8f; 2) a hydrogen bond linking Phe-8f NH to Lys-97 O;3) a salt link between Glu-11f and Arg-173; 4) two antiparallel beta-sheet hydrogen bonds between Gly-14f and Gly-216; and 5) the insertion of Arg-16f into the specificity pocket. Binding of the peptide is accompanied by a considerable shift in two of the loops near the active site relative to human D-phenyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK)-thrombin.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Structural changes that accompany the reduced catalytic efficiency of two semisynthetic ribonuclease analogs.

The structures of two catalytically defective semi-synthetic RNases obtained by replacing aspartic acid 121 with asparagine or alanine have been determined and refined at a resolution of 2.0 A (R = 0.186 and 0.172, respectively). When these structures are compared with the refined 1.8-A structure (R = 0.204) of the fully active aspartic acid-containing enzyme (Martin, P.D., Doscher, M.S., and Edwards, B. F. P. (1987) J. Biol. Chem. 262, 15930-15938), numerous and widespread changes, much greater in number and magnitude than the small structural variations noted previously between the semisynthetic complex and RNase A, are found to have occurred. These changes include the movement of the loop containing residues 65-72 away from the active site, a more or less generalized relocation of crystallographically bound water molecules, and a number of rearrangements in the hydrogen bonding network at the active site. Most changes are far removed from the immediate site of the modifications and are distributed essentially throughout the molecule. The details of many of these changes are unique to each analog. In the asparagine analog, a destabilization in the positioning of active site residue His-119 also appears to have occurred.

Alanine↗
PubMed1992

A model of the platelet factor 4 complex with heparin.

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (1----4)-O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1----4)-(2-deoxy-2-sulfamino-2-D-glucopyranosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 A resolution with R = 0.237. Each monomer of BPF4 contains an alpha-helix lying across 3 strands of antiparallel beta-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the alpha-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer.

Amino Acid Sequence↗
PubMed1991

Refined crystal structure of ytterbium-substituted carp parvalbumin 4.25 at 1.5 A, and its comparison with the native and cadmium-substituted structures.

The crystal structure of carp parvalbumin 4.25 containing a 1:1 molar ratio of ytterbium chloride to protein has been refined at 1.5 A resolution by restrained least-squares methods to a crystallographic R value of 0.199. The crystal structure confirms the NMR studies, which suggest that low concentrations of ytterbium cause an extensive displacement of calcium from the EF metal binding site. A comparison of the ytterbium-substituted model with the native and cadmium-substituted structure show no significant differences, except around the substituted EF metal-binding region. The displacement of calcium by ytterbium at the EF site has caused a movement in the polypeptide backbone of Ser-91 and Asp-92. This movement resulted in an increase in the number of oxygen ligands bound to ytterbium in the EF site from seven to eight.

Amino Acid Sequence↗
PubMed1991

Crystal structure of interleukin 8: symbiosis of NMR and crystallography.

The crystal structure of a host defense system chemotactic factor, interleukin 8, has been solved by molecular replacement using as a model the solution structure derived from nuclear magnetic resonance experiments. The structure was refined with 2 A x-ray data to an R factor of 0.187 (0.217 at 1.6 A). A comparison indicates some potential differences between the structure in solution and in the crystalline state. Our analysis also predicts that residues 4 through 9 on the amino terminus and the beta-bend, which includes His-33, may be important for receptor binding.

Amino Acid Sequence↗
PubMed1990

Refined crystal structure of calcium-liganded carp parvalbumin 4.25 at 1.5-A resolution.

The crystal structure of carp parvalbumin (pI = 4.25) has been refined by restrained least-squares analysis employing X-ray diffractometer data to 1.5-A resolution. The final residual for 12,653 reflections between 10 and 1.5 A with I(hkl) greater than 2 sigma(I) is 0.215. A total of 74 solvent molecules were included in the least-squares analysis. The root mean square deviation from ideality of bond lengths is 0.024 A. The model has a root mean square difference of 0.59 A from the positions of the main-chain atoms in a previously reported structure [Moews, P. C., & Kretsinger, R. H. (1975) J. Mol. Biol. 91, 201-228], which was refined by difference Fourier syntheses using data collected by film to 1.9 A. Although the overall features of the two models are very similar, there are significant differences in the amino-terminal region, which was extensively refit, and in the number of oxygen atoms liganding calcium in the CD and EF sites, which increased from six to seven in the CD site and decreased from eight to seven in the EF site.

Animals↗