PubMed Health⌕ Search

Biomedical subjects

W Pangborn

Publications and source records attributed to W Pangborn.

36 records · Page 2Linked to original sources
PubMed1996

Studies on the three-dimensional structure of estrogenic 17 beta-hydroxysteroid dehydrogenase.

The structure-function relationship of the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), a pivotal enzyme in the synthesis of active sex hormones, has been studied via protein chemistry and crystallography. A highly active and homogeneous 17 beta-HSD1 was prepared with a rapid purification from human placenta. We then characterized the native and expressed enzyme, and concluded, for the first time, that 17 beta-HSD1 is formed by two identical subunits. The enzyme was also overproduced in insect cells with a baculovirus expression system. The highly active 17 beta-HSD1 preparation was successfully crystallized in the presence of NADP-, polyethylene glycol, beta-octylglucoside and glycerol, resulting in the first diffraction quality crystals of any steroid-converting enzyme from a human source. The three-dimensional structure of 17 beta-HSD1 was determined at 2.2 A resolution, showing that the overall structure of the enzyme is similar to the other enzymes in the short-chain dehydrogenase family, with a conserved Tyr-X-X-X-Lys sequence and a serine residue in the active site. It is distinguished from the other known structures reported for short-chain dehydrogenases by the insertion of two helix-turn-helix motifs that appear to govern membrane association and substrate specificity [corrected]. More recently, the complex of 17 beta-HSD1 with estradiol has been successfully crystallized and its structure determined. The latter demonstrates detailed information of the interactions between the substrate and residues Ser142, Tyr155, His221 and Glu282 of the enzyme. These interactions and the complementarity of the substrate with the hydrophobic binding pocket make critical contributions to the enzyme specificity. The above results provide a strong basis for the design of potent inhibitors of this pivotal steroid dehydrogenase.

Estradiol Dehydrogenases↗
PubMed1995

Structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution.

BACKGROUND: The principal human estrogen, 17 beta-estradiol, is a potent stimulator of certain endocrine-dependent forms of breast cancer. Because human estrogenic 17 beta-hydroxysteroid dehydrogenase (type I 17 beta-HSD) catalyzes the last step in the biosynthesis of 17 beta-estradiol from the less potent estrogen, estrone, it is an attractive target for the design of inhibitors of estrogen production and tumor growth. This human enzyme shares less than 15% sequence identity with a bacterial 3 alpha,20 beta-HSD, for which the three-dimensional structure is known. The amino acid sequence of 17 beta-HSD also differs from that of bacterial 3 alpha,20 beta-HSD by two insertions (of 11 and 14 residues) and 52 additional residues at the C terminus. RESULTS: The 2.20 A resolution structure of type I 17 beta-HSD, the first mammalian steroidogenic enzyme studied by X-ray crystallographic techniques, reveals a fold characteristic of the short-chain dehydrogenases. The active site contains a Tyr-X-X-X-Lys sequence (where X is any amino acid) and a serine residue, features that are conserved in short-chain steroid dehydrogenases. The structure also contains three alpha-helices and a helix-turn-helix motif, not observed in short-chain dehydrogenase structures reported previously. No cofactor density could be located. CONCLUSIONS: The helices present in 17 beta-HSD that were not in the two previous short-chain dehydrogenase structures are located at one end of the substrate-binding cleft away from the catalytic triad. These helices restrict access to the active site and appear to influence substrate specificity. Modeling the position of estradiol in the active site suggests that a histidine side chain may play a critical role in substrate recognition. One or more of these helices may also be involved in the reported association of the enzyme with membranes. A model for steroid and cofactor binding as well as for the estrone to estradiol transition state is proposed. The structure of the active site provides a rational basis for designing more specific inhibitors of this breast cancer associated enzyme.

Amino Acid Sequence↗
PubMed1995

Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase.

BACKGROUND: Candida cylindracea cholesterol esterase (CE) reversibly hydrolyzes cholesteryl linoleate and oleate. CE belongs to the same alpha/beta hydrolase superfamily as triacylglycerol acyl hydrolases and cholinesterases. Other members of the family that have been studied by X-ray crystallography include Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase. CE is homologous to C. rugosa lipase 1, a triacylglycerol acyl hydrolase, with which it shares 89% sequence identity. The present study explores the details of dimer formation of CE and the basis for its substrate specificity. RESULTS: The structures of uncomplexed and linoleate-bound CE determined at 1.9 A and 2.0 A resolution, respectively, reveal a dimeric association of monomers in which two active-site gorges face each other, shielding hydrophobic surfaces from the aqueous environment. The fatty-acid chain is buried in a deep hydrophobic pocket near the active site. The positioning of the cholesteryl moiety of the substrate is equivocal, but could be modeled in the hydrophobic core of the dimer interface. CONCLUSIONS: The monomer structure is the same in both the complexed and uncomplexed crystal forms. The dimers differ in the relative positions of the two monomers at the dimer interface. Of the 55 residues that are different in CE from those in C. rugosa lipase 1, 23 are located in the active site and at the dimer interface. The altered substrate specificity is a direct consequence of these substitutions.

Animals↗
PubMed1995

Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

Amino Acid Sequence↗
PubMed1994

Mechanism of inhibition of 3 alpha, 20 beta-hydroxysteroid dehydrogenase by a licorice-derived steroidal inhibitor.

BACKGROUND: Bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD) reversibly oxidizes the 3 alpha and 20 beta hydroxyl groups of androstanes and pregnanes and uses nicotinamide adenine dinucleotide as a cofactor. 3 alpha, 20 beta-HSD belongs to a family of short-chain dehydrogenases that has a highly conserved Tyr-X-X-X-Lys sequence. The family includes mammalian enzymes involved in hypertension, digestion, fertility and spermatogenesis. Several members of the enzyme family, including 3 alpha, 20 beta-HSD, are competitively inhibited by glycyrrhizic acid, a steroidal compound found in licorice, and its derivative, carbenoxolone, an anti-inflammatory glucocorticoid. RESULTS: The three-dimensional structure of the enzyme-carbenoxolone complex has been determined and refined at 2.2 A resolution to a crystallographic R-factor of 19.4%. The hemisuccinate side chain of carbenoxolone makes a hydrogen bond with the hydroxyl group of the conserved residue Tyr152 and occupies the position of the nicotinamide ring of the cofactor. The occupancies of the inhibitor in four independent catalytic sites refine to 100%, 95%, 54% and 36%. CONCLUSIONS: The steroid binds at the catalytic site in a mode much like the previously proposed mode of binding of the substrate cortisone. No bound cofactor molecules were found. The varying occupancy of steroid molecules observed in the four catalytic sites is either due to packing differences or indicates a cooperative effect among the four sites. The observed binding accounts for the inhibition of 3 alpha, 20 beta-HSD.

Binding Sites↗
PubMed1994

Methotrexate-resistant variants of human dihydrofolate reductase. Effects of Phe31 substitutions.

Substitution of glycine or alanine for phenylalanine 31 in human dihydrofolate reductase produces variants that are inhibited less by methotrexate (MTX) than the previously reported serine variant. The 100 times decrease in MTX affinity for the glycine variant is due to slower binding, and to inability of the initial complex to isomerize to a nondissociating conformer. A polar group at position 31 is unnecessary for resistance, but residues larger than serine confer no resistance. The glycine variant best fulfills criteria for gene therapy: low Km for H2folate, high kcat, and good stability. Although kcat is unaltered by these mutations, the rate of hydride transfer is greatly decreased. Presteady-state measurements have enabled a complete catalytic scheme to be constructed for the glycine variant that predicts observed steady-state behavior. The crystal structures of inhibitor complexes of the serine, alanine, and glycine mutants and of the wild-type enzyme show that the mutations cause little perturbation of the protein backbone, of side chains of residues at the active site, or of the bound inhibitor. A molecule of bound water occupies the space vacated by the phenyl group.

Amino Acid Sequence↗
PubMed1994

Structural determination and packing analysis of a cholesteryl caprate/cholesteryl laurate solid solution.

This paper describes the X-ray crystal structure analysis of a cholesteryl ester solid solution, cholesteryl decanoate/cholesteryl laurate, grown from a bulk concentration with molar ratio 0.56/0.43. The unit cell is monoclinic with a = 12.969, b = 9.048, c = 31.137 A, and beta = 91.12 degrees and the space group P2(1) with Z = 4 (two molecules per asymmetric unit). The cell constants closely represent an average value of crystal parameters for the two pure components (hence, nearly corresponding to Vegard's law). Although the overall monolayer 1 lamellar packing is superficially similar to the earlier-studied cholesteryl undecanoate/cholesteryl laurate solid solution, a more partitioned distribution of acyl chains, i.e., a microfractionation corresponding to the observed nonideal phase behavior, is suggested. The behavior is similar to that found for n-paraffin binaries cooled below a binodal phase boundary. Although it cannot be detected conclusively in this determination (due to high thermal motion of terminal acyl chain atoms), the non-stoichiometric combination of components also requires some partial occupancy of atomic sites on the chain termini. This structural arrangement is contrasted with the alternative expression found earlier for the more ideal undecanoate/laurate solid solution, i.e., random co-packing leading to fractional atomic occupancy in an average laurate structure. The final weighted R factor for 4578 reflections is 0.138.

Cholesterol Esters↗
PubMed1993

Crystallization and preliminary X-ray diffraction analysis of the complex of human placental 17 beta-hydroxysteroid dehydrogenase with NADP+.

Single crystals of human placental 17 beta-hydroxysteroid dehydrogenase, an enzyme that plays an important role in the interconversion of estrogens, were obtained as the NADP+ complex. These are the first crystals suitable for complete X-ray structure analysis ever reported for a steroid-converting enzyme from a human source. The crystals were grown by vapor diffusion at pH 7.5 with polyethyleneglycol (4000) as the precipitating agent. They have a monoclinic space group C2 and unit cell parameters are a = 123.03 A, b = 45.03 A, c = 61.29 A, and beta = 99.1 degrees. A complete set of diffraction data to 2.9 A has been collected on native crystals.

17-Hydroxysteroid Dehydrogenases↗
PubMed1993

Crystallization and preliminary X-ray diffraction studies of a mammalian steroid dehydrogenase.

20 beta-Hydroxysteroid dehydrogenase from the cytosolic fraction of neonatal pig testis is a NADPH-dependent enzyme that catalyzes the reduction of the C-20 ketone of C21-steroids. It is 85% homologous in amino acid sequence to the human enzyme, carbonyl reductase. The enzyme has been crystallized from 36% saturated ammonium sulfate in 10 mM 2-[N-Morpholino]ethanesulfonic acid buffer. The size and the quality of nicely formed square bi-pyramidal crystals were improved by using a "seeding" technique. The crystals diffract X-rays to at least 2.5 A resolution. The space group is P4(1)2(1)2 (or P4(3)2(1)2) and the unit-cell dimensions are a = b = 58.53 A, c = 165.64 A. There is one molecule (M(r) = 30.5 kDa; 289 amino acid residues) in the asymmetric unit. An intensity data set to 2.5 A has been collected with an overall Rmerge of 6.6% for all reflections.

Animals↗
PubMed1992

Purification and crystallization of Lactobacillus casei folylpolyglutamate synthetase expressed in Escherichia coli.

Folylpolyglutamate synthetase (FPGS) from Lactobacillus casei has been crystallized with polyethylene glycol and acetate buffer at pH 5.0. The enzyme was obtained from Escherichia coli strain SF4 harboring the L. casei FPGS chromosomal gene on a pEMBL vector (pGT3-8.1). Crystals of the enzyme were obtained which diffract to 2.6 A resolution. The crystals are monoclinic, space group P2(1), with unit cell dimensions of a = 54.07 A, b = 45.83 A, c = 84.37 A and beta = 107.92 degrees. A unit cell contains one molecule of the 43,000 Da enzyme per asymmetric unit. A complete X-ray data set on the native crystals has been collected.

Escherichia coli↗
PubMed1992

Absolute configuration and conformation of the pure opioid antagonist (+)-2,9 alpha-dimethyl-5-(m-hydroxyphenyl)morphan.

(+)-2,9 alpha-Dimethyl-5-(m-hydroxyphenyl)morphan is the only phenylmorphan analog whose affinity for opioid kappa-receptors is greater than its affinity for opioid mu-receptors. Pharmacologically, the compound is a pure opioid antagonist devoid of agonist activity in in vivo assays of antinociception. The absolute configuration of the compound has been determined to be (1R,5S,9R) from an X-ray crystallographic study of the chloride salt. Thus, the absolute configuration corresponds to that of the atypical opioid agonist (-)-phenylmorphan while the weak atypical agonist (-)-2,9 alpha-dimethyl-5-(m- hydroxyphenyl)morphan corresponds to the potent morphine-like (+)-phenylmorphan. The preferred orientations of the phenyl ring for the two stereoisomers were determined using the molecular mechanics program MM2-87 and found to vary from that of the two parent compounds. The atypical properties of the two 9 alpha-methyl analogs is consistent with an opioid ligand model which proposes that morphine-like properties require a particular range of phenyl orientations. There was good agreement between the structure obtained from X-ray crystallography and computed with the MM2-87 program.

Chemical Phenomena↗
PubMed1992

Purification and crystallization of insecticidal delta-endotoxin CryIIIB2 from Bacillus thuringiensis.

CryIIIB2, an insecticidal protein from Bacillus thuringiensis has been crystallized from 0.6 M NaBr and HEPES buffer at pH 7.0 and X-ray diffraction data collected on a native crystal to 2.4 A. The insecticidal protein was obtained from a Bacillus thuringiensis (Bt) strain EG7231. Crystals of the endotoxin are orthorhombic, space group C2221, with unit cell dimensions of a = 122.44, b = 131.81, and c = 105.37 A. A unit cell contains one molecule of the 67,000 Da endotoxin per asymmetric unit.

Bacillus thuringiensis↗
PubMed1992

Inhibition of Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase by licorice-derived compounds and crystallization of an enzyme-cofactor-inhibitor complex.

Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase reduces the C20 ketone on glucocorticoids and progestins. We find that two licorice-derived compounds, glycyrrhizic acid and carbenoxolone, inhibit this enzyme with microM Kis. Inhibition is competitive, indicating that these compounds are binding at or close to the catalytic site. Carbenoxolone's high aqueous solubility and affinity for 3 alpha,20 beta-hydroxysteroid dehydrogenase enabled us to prepare crystals of a carbenoxolone-NADH-enzyme ternary complex, which preliminary X-ray analysis indicates has a crystal structure that is significantly different from that of the 3 alpha,20 beta-hydroxysteroid dehydrogenase-NADH complex. A comparison of the tertiary structures of these two complexes should prove useful in understanding this enzyme's catalytic mechanism, as well as those of two homologous enzymes, mammalian 11 beta-hydroxysteroid dehydrogenase and 15-hydroxyprostaglandin dehydrogenase that also are inhibited by carbenoxolone.

Binding Sites↗
PubMed1979

Studies on the mechanism of membrane fusion: evidence for an intermembrane Ca2+-phospholipid complex, synergism with Mg2+, and inhibition by spectrin.

The interaction of Ca2+ and Mg2+ with phosphatidylserine (PS) vesicles in 0.1 M NaCl aqueous solution was studied by equilibrium dialysis binding, X-ray diffraction, batch microcalorimetry, kinetics of cation-induced vesicle aggregation, release of vesicle contents, and fusion. Addition of either cation causes aggregation of PS vesicles and produces complexes with similar stoichiometry (1:2 cation/PS) at saturating concentrations, although the details of the interactions and the resulting complexes are quite different. Addition of Ca2+ to PS vesicles at T greater than or equal to 25 degrees C induces the formation of an "anhydrous" complex of closely apposed membranes with highly ordered crystalline acyl chains and a very high transition temperature (Tc greater than 100 degrees C). The formation of this complex is accompanied by a release of heat (5.5 kcal/mol), rapid release of vesicle contents, and fusion of the vesicles into larger membranous structures. By contrast, addition of Mg2+ produces a complex with PS which is much more hydrated, has no crystallization of the acyl chains at T greater than or equal to 20 degrees C, and has comparatively little fusion. Studies with both Ca2+ and Mg2+ added simultaneously indicate that there is a synergistic effect between the two cations, which results in an enhancement of the ability of Ca2+ to form its specific complex with PS at lower concentrations. The presence of the erythrocyte protein "spectrin" inhibits this synergism and interferes with the formation of the specific PS/Ca complex. It also inhibits the fusion of PS vesicles. It is proposed that the unique PS/Ca complex, which involves close apposition of vesicle membranes, is an intermembrane "trans" complex. We further propose that such a complex is a key step for the resultant phase transition and fusion of PS vesicles. By contrast, the PS/Mg complex is proposed to be a "cis" complex with respect to each membrane. The results are discussed in terms of the mechanism of membrane fusion.

Calcium↗
PubMed1978

Specificity of Ca2+ and Mg2+ binding to phosphatidylserine vesicles and resultant phase changes of bilayer membrane structure.

Differences in the interaction of Ca2+ and Mg2+ with phosphatidylserine vesicles were revealed by binding studies, differential scanning calorimetry and X-ray diffraction. The two cations produced structurally different complexes with phosphatidylserine as evidenced by phase transition characteristics, lamellar spacings, and hydrocarbon packing. Ca2+ effectively completes with Mg2+ for phosphatidylserine binding sites. Analysis of the binding data showed that Ca2+ had a ten-fold greater intrinsic binding constant for phosphatidylserine.

Binding Sites↗
PubMedDate not supplied

[A new crystal form of the Fab fragment of a monoclonal antibody to human interleukin-2: the three-dimensional structure at 2.7 A resolution].

The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 in a new crystal form (space group P2(1)2(1)2(1); unit cell parameters: a = 42.82, b = 90.68, and c = 139.82 A) was determined by the X-ray molecular replacement method at the resolution of 2.7 A. The protein folding and the stereochemistry of its antigen-binding site were comparatively analyzed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

Antibodies, Monoclonal↗