Biomedical subjects

B Stec

Publications and source records attributed to B Stec.

36 records · Page 2Linked to original sources

PubMed1997

Structure of human plasminogen kringle 4 at 1.68 a and 277 K. A possible structural role of disordered residues.

Despite considerable effort to elucidate the functional role of the kringle domains, relatively little is known about interactions with other protein domains. Most of the crystal structures describe the interactions at the kringle active site. This study suggests a novel way to interpret structural results such as disorder located away from an active site. The crystal structure of human plasminogen kringle 4 (PGK4) has been refined against 10-1.68 A resolution X-ray data (R(merge) = 3.7%) to the standard crystallographic R = 14.7% using the program X-PLOR. The crystals of PGK4 showed significant instability in cell dimensions (changes more than 1.5 A) even at 277 K. The refinement revealed structural details not observed before [Mulichak, Tulinsky & Ravichandran (1991). Biochemistry, 30, 10576-10588], such as clear density for additional side chains and more extensive disorder. Discrete disorder was detected for residues S73, S78, T80, S89, S91, S92, Ml12, S132, C138 and K142. Most of the disordered residues form two patches on the surface of the protein. This localized disorder suggests that these residues may play a role in quaternary interactions and possibly form an interface with the other domains of proteins that contain kringles, such as plasminogen. Although, an additional residue D65 was refined at the beginning of the sequence, still more residues near the peptide cleavage site must be disordered in the crystal.

Journal Article↗

PubMed1997

The road less travelled: taming phosphatases.

Phosphatases are important in signal transduction, bacterial pathogenesis and several human diseases. So far, however, it is their opposite numbers, the kinases, that have received more attention from chemists. Recent progress in inhibitor development offers hope that new probes of cellular processes, and perhaps novel therapeutic agents, may soon become available.

Affinity Labels↗

PubMed1997

Trapping and visualization of a covalent enzyme-phosphate intermediate.

Using a mutant version of E. coli alkaline phosphatase, we succeeded in trapping and determining the structure of the phospho-enzyme intermediate. The X-ray structure also revealed the catalytic water molecule, bound to one of the active site zinc ions, positioned ideally for the apical attack necessary for the hydrolysis of the intermediate.

Alkaline Phosphatase↗

PubMed1996

Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution.

Lipoxygenases, which are widely distributed among plant and animal species, are Fe-containing dioxygenases that act on lipids containing (Z,Z)-pentadiene moieties in the synthesis of compounds with a variety of functions. Utilizing an improved strategy of data collection, low temperature, and synchrotron radiation of short wavelength, the structure of ferrous soybean lipoxygenase L-1, a single chain protein of 839 amino acid residues, has been determined by X-ray crystallography to a resolution of 1.4 A. The R-factor for the refined model is 19.7%. General features of the protein structure were found to be consistent with the results of prior crystallographic studies at lower (2.6 A) resolution. In contrast to the prior studies, the binding of a water molecule to the active site Fe was established. The octahedral coordination sphere of the Fe also includes the side chains of His499, His504, His690, and Asn694 as well as the terminal carboxylate of Ile839, which binds as a monodentate ligand. Asn694 is involved in a number of labile polar interactions with other protein groups, including an amide-aromatic hydrogen bond, and appears to be a weak ligand. Several possible access routes for dioxygen and fatty acids to the internal active site and substrate binding cavity are described. The protein structure restricts access to the Fe site such that the formation of an organo-Fe intermediate seems improbable. Structural restrictions pertinent to other proposed reaction intermediates, such as planar pentadienyl and nonplanar allyl radicals, are also discussed.

Crystallography, X-Ray↗

PubMed1996

Crystal structures of the active site mutant (Arg-243-->Ala) in the T and R allosteric states of pig kidney fructose-1,6-bisphosphatase expressed in Escherichia coli.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-1,6-bisphosphate was reduced 10-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243-->Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.

Adenosine Monophosphate↗

PubMed1996

Evidence for an active T-state pig kidney fructose 1,6-bisphosphatase: interface residue Lys-42 is important for allosteric inhibition and AMP cooperativity.

During the R-->T transition in the tetrameric pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412-4420). The change in quaternary structure involving the rotation of the upper dimer by 17 degrees relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys-42 is involved in a complex set of intersubunit interactions across the dimer-dimer interface with residues of the 190's loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys-42, we have replaced it with alanine using site-specific mutagenesis. The kcat and K(m) values for Lys-42-->Ala Fru-1,6-P2ase were 11 s-1 and 3.3 microM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys-42-->Ala Fru-1,6-P2ase was similar kinetically in terms of K(m) and kcat, the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high- and low-affinity binding sites. Saturation of the high-affinity sites only reduced the activity by 30%, compared to 100% for the wild-type enzyme. In order to determine in what structural state the enzyme was after saturation of the high-affinity sites, the Lys-42-->Ala enzyme was crystallized in the presence of Mn2+, fructose-6-phosphate (Fru-6-P), and 100 microM AMP and the data collected to 2.3 A resolution. The X-ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru-6-P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys-42 and residues of the 190's loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimer-dimer interface as opposed to the monomer-monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.

Adenosine Monophosphate↗

PubMed1996

Expression, purification and characterization of recombinant crambin.

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues), has been successfully expressed in Escherichia coli from an artificial, synthetic gene. Several expression systems were investigated. Ultimately, crambin was successfully expressed as a fusion protein with the maltose binding protein, which was purified by affinity chromatography. Crambin expressed as a C-terminal domain was then cleaved from the fusion protein with Factor Xa protease and purified. Circular dichroism spectroscopy and amino acid analysis suggested that the purified material was identical to crambin isolated from seed. For positive identification the protein was crystallized from an ethanol-water solution, by a novel method involving the inclusion of phospholipids in the crystallization buffer, and then subjected to crystallographic analysis. Diffraction data were collected at the Brookhaven synchrotron (beamline-X12C) to a resolution of 1.32 A at 150 K. The structure, refined to an R value of 9.6%, confirmed that the cloned protein was crambin. The availability of cloned crambin will allow site-specific mutagenesis studies to be performed on the protein known to the highest resolution.

ATP-Binding Cassette Transporters↗

PubMed1995

Refinement of purothionins reveals solute particles important for lattice formation and toxicity. Part 1: alpha1-purothionin revisited.

The three-dimensional structure of alpha(1)-purothionin (alpha(1)-PT), a wheat-germ protein and a basic lytic toxin, was previously solved by molecular-replacement methods using an energy-minimized predicted model and refined to an R-factor of 21.6% [Teeter, Ma, Rao & Whitlow (1990). Proteins Struct. Funct. Genet. 8, 118-1321. Some deficiencies of the model motivated us to revisit the structure and to continue the refinement. Here we report a significantly improved structure refined to an R-factor of 15.5% with excellent geometry. The refinement of this relatively low resolution structure ( approximately 2.8 A) is well suited to test the limitations of classical methods of refinement and to address the problem of overfitting, The final structure contains 434 atoms including 330 protein atoms, 70 waters, three acetates, two glycerols, one sec-butanol and one phosphate. The key solute molecules (acetate ion and phosphate ion) play a crucial role in the lattice formation. Phosphate and glycerol found in the structure may be important for biological activity of the toxins.

Journal Article↗

PubMed1995

Refinement of purothionins reveals solute particles important for lattice formation and toxicity. Part 2: structure of beta-purothionin at 1.7 A resolution.

The crystal structure of beta-purothionin (beta-PT) has been determined at 1.7 A resolution. beta-PT and previously solved alpha(l)-PT belong to a family of membrane-active plant toxins homologous to crambin. (beta-PT crystallizes in the same space group as alpha(l)-PT (1422) but with the c axis 3 A longer than (alpha(l)-PT. The unit-cell dimensions of beta-PT crystals are a = b = 53.94 and c = 72.75 A. Two data sets were collected on a multiwire area detector, each with R(sym) around 6.0%, and were merged to get a single data set at 1.7 A, (R(merge) = 9.6%). The X-ray structure of alpha(l)-PT was used to build a starting model for beta-PT. The beta-PT model was refined using the program PROLSQ from 10 to 1.7 A resolution to an R-factor of 19.8% with very good geometry. The final structure contains 439 atoms including 337 protein atoms, 77 waters, two acetates, two glycerols and one phosphate. The high-resolution structure of the beta-PT agreed well with that of the lower resolution alpha(l)-PT structure only after the latter was extensively rerefined. Both refinements revealed phosphate and glycerol molecules which are important in lattice formation. The binding of phosphate and glycerol molecules to purothionins (PT) was confirmed by NMR and was implicated in the biological activity of toxins. Modeling of phospholipid binding to PT based on glycerol and phosphate-binding site could shed light on the lytic toxicity of this protein-toxin family. Although the structures of (alpha(l)-PT and beta-PT preserve the overall fold of crambin, they exhibit key differences that are directly relevant to the toxicity of thionins.

Journal Article↗

PubMed1995

Full-matrix refinement of the protein crambin at 0.83 A and 130 K.

This paper describes the first successful full-matrix least-squares (FMLS) refinement of a protein structure. The example used is crambin which is a small hydrophobic protein (4.7 kDa, 46 residues). It proves the feasibility of refining such large molecules by this classic method, routinely applied to small molecules. The final structure with 381 non-H protein atoms (54 protein atoms in alternative positions), 367 H atoms, 162 water molecules (combined occupancy 93) and one disordered ethanol molecule converged to a standard unweighted crystallographic R-factor of R = 9.0% when refined against F with reflections stronger than F > 2sigma(F) and R = 9.5% when refined against F(2). The programs RFINE [Finger & Prince (1975). Natl Bur. Stand. (US) Tech. Note 854. A System of Fortran IV Computer Programs for Crystal Structure Computations] and SHELXL93 [Sheldrick (1993). SHELXL93. Program for Crystal Structure Refinement, Univ. of Göttingen, Germany] were used for FMLS refinement with the high-resolution low-temperature (0.83 A, 130 K) data set of a mixed-sequence form of crambin. A detailed analysis of the models obtained in FMLS and PROLSQ [restrained least squares or RLS; Teeter, Roe & Heo (1993). J. Mol. Biol. 230, 292-311] refinements with the same data set is presented. The differences between the models obtained by both FMLS and RLS refinements are systematic but negligible and advantages and shortcomings of both methods are discussed. The final structure has very good geometry, fully comparable to the geometry of other structures in this resolution range. Ideal values used in PROLSQ and those by Engh & Huber [Engh & Huber (1991). Acta Cryst. A47, 392-400] differ significantly from this refinement and we recommend a new standard. FMLS refinement constitutes a sensitive tool to detect and model disorder in highly refined protein structures. We describe the modeling of temperature factors by the TLS method [Schomaker & Trueblood (1968). Acta Cryst. B24, 63-76]. Rigid body-TLS refinements led to a better understanding of different modes of vibrations of the molecule. Refinements using F(2) or F protocols converged and reached slightly different minima. Despite theoretical support for F(2)-based refinement, we recommend refinement on structure factors.

Journal Article↗

PubMed1994

Homology modeling of the dopamine D2 receptor and its testing by docking of agonists and tricyclic antagonists.

We present the first model of dopamine D2 receptor transmembrane helices constructed directly from the bacteriorhodopsin (bR) coordinates derived from two-dimensional electron diffraction experiments. We have tested this model by its ability to accommodate rigid agonist and semirigid antagonist molecules which were docked into the putative binding pocket with stabilizing interactions. The model is consistent with structure-activity relationships of agonists and antagonists that interact with the receptor. It also illuminates data on a Na+ site for regulation of receptor function. The plausibility of the model is increased by its consistency with many mutagenesis studies on G protein-coupled receptors. Further, this model provides a basis to suggest testable molecular mechanisms for changes in the D2 conformational states for high- and low-affinity binding and signal transduction. Changes in the conformational state of the receptor are hypothesized to be due partly to movement of helix 7. In contrast to the model presented here, other published models were built using ideal helical structures or following the sense of the bacteriorhodopsin structure rather than the actual available coordinates. The presented model for the dopamine G protein-coupled receptor can be reconciled with the recent rhodopsin projection structure (Schertler, G. F. X.; Villa, C.; Henderson, R. Projection Structure of Rhodopsin.

Allosteric Regulation↗

PubMed1991

Mechanism of enolase: the crystal structure of enolase-Mg2(+)-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-A resolution.

Enolase in the presence of Mg2+ catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) and the reverse reaction, the hydration of PEP to PGA. The structure of the ternary complex yeast enolase-Mg2(+)-PGA/PEP has been determined by X-ray diffraction and refined by crystallographic restrained least-squares to an R = 16.9% for those data with I/sigma (I) greater than or equal to 2 to 2.2-A resolution with a good geometry of the model. The structure indicates the substrate molecule in the active site has its hydroxyl group coordinated to the Mg2+ ion. The carboxylic group interacts with the side chains of His373 and Lys396. The phosphate group is H-bonded to the guanidinium group of Arg374. A water molecule H-bonded to the carboxylic groups of Glu168 and Glu211 is located at a 2.6-A distance from carbon-2 of the substrate in the direction of its proton. We propose that this cluster functions as the base abstracting the proton in the catalytic process. The proton is probably transferred, first to the water molecule, then to Glu168, and further to the substrate hydroxyl to form a water molecule. Some analogy is apparent between the initial stages of the enolase reverse reaction, the hydration of PEP, and the proteolytic mechanism of the metallohydrolases carboxypeptidase A and thermolysin. The substrate/product binding is accompanied by large movements of loops Ser36-His43 and Ser158-Gly162. The role of these conformational changes is not clear at this time.

Amino Acid Sequence↗

PubMed1991

Inhibition of enolase: the crystal structures of enolase-Ca2(+)- 2-phosphoglycerate and enolase-Zn2(+)-phosphoglycolate complexes at 2.2-A resolution.

Enolase is a metalloenzyme which catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP). Mg2+ and Zn2+ are cofactors which strongly bind and activate the enzyme. Ca2+ also binds strongly but does not produce activity. Phosphoglycolate (PG) is a competitive inhibitor of enolase. The structures of two inhibitory ternary complexes: yeast enolase-Ca2(+)-PGA and yeast enolase-Zn2(+)-PG, were determined by X-ray diffraction to 2.2-A resolution and were refined by crystallographic least-squares to R = 14.8% and 15.7%, respectively, with good geometries of the models. These structures are compared with the structure of the precatalytic ternary complex enolase-Mg2(+)-PGA/PEP (Lebioda & Stec, 1991). In the complex enolase-Ca2(+)-PGA, the PGA molecule coordinates to the Ca2+ ion with the hydroxyl group, as in the precatalytic complex. The conformation of the PGA molecule is however different. In the active complex, the organic part of the PGA molecule is planar, similar to the product. In the inhibitory complex, the carboxylic group is in an orthonormal conformation. In the inhibitory complex enolase-Zn2(+)-PG, the PG molecule coordinates with the carboxylic group in a monodentate mode. In both inhibitory complexes, the conformational changes in flexible loops, which were observed in the precatalytic complex, do not take place. The lack of catalytic metal ion binding suggests that these conformational changes are necessary for the formation of the catalytic metal ion binding site.

Amino Acid Sequence↗

PubMed1991

Mapping of isozymic differences in enolase.

The existence of the isozymes of non-regulatory enzymes often has been linked to their interaction with other macromolecules. Enolase, a non-regulatory enzyme, has three isozymes for which sequences have been determined in two or more vertebrate species. The positions in the enolase sequences that differ between the isozymes were mapped in the 3-D structure of the enzyme. The positions in a given isozymic form which were not conserved in different species were considered to be resulting from the neutral drift of sequences and rejected. Also, the residues with no accessible surface were rejected. Three areas with relatively high densities of isozymic substitutions were found. We consider them as the likely sites of contact with other macromolecules.

Amino Acid Sequence↗

PubMed1990

Refined structure of yeast apo-enolase at 2.25 A resolution.

The crystal structure of apo-enolase from baker's yeast (Saccharomyces cerevisiae) was established at 2.25 A resolution using a restrained least-squares refinement method. Based on 21,077 independent reflections of better than 8 A resolution, a final R-factor of 15.4% was obtained with a model obeying standard geometry within 0.017 A in bond length and 3.5 degrees in bond angles. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.18 A. The refinement confirmed the heterodox, non-parallel character of the 8-fold beta alpha-barrel domain with beta beta alpha alpha(beta alpha)6 topology. The reported structure for which the data were collected at pH 5.0 represents an apo-form of the enzyme. Of the three carboxylic ligands that form the conformational metal ion binding site two, Glu295 and Asp320, are very close and presumably form a strong acidic type hydrogen bond with the proton partially replacing the electric charge of the physiological cofactor Mg2+. The single sulfate ion found in the structure is in the active site cavity, co-ordinated to the side-chains of Lys345 and Arg374, and to the N atom of Ser375. It is located about 7.4 A from the conformational metal ion binding site. It occupies the site in which the phosphate group of the substrate binds.

Amino Acid Sequence↗

PubMed1990

Crystallization of alcohol oxidase from Pichia pastoris. Secondary structure predictions indicate a domain with the eightfold beta/alpha-barrel fold.

Alcohol oxidase from Pichia pastoris has been crystallized from polyethylene glycol 4000 solutions. The crystals are tetragonal, a = 228 A, c = 456 A space group P4(1)2(1)2. The crystals scatter only to about 6 A resolution; their poor crystallinity may have some physiological function. Secondary structure predictions suggest that the C-terminal part of the molecule, residues 311-664, has the folding of an eightfold beta/alpha-barrel (TIM barrel). This would indicate common ancestry with four other flavoenzymes: canavalin, glycolate oxidase, flavocytochrome b, and trimethylamine dehydrogenase.

Alcohol Oxidoreductases↗

PubMed1989

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology.

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.

Binding Sites↗

PubMed1988

Crystal structure of enolase indicates that enolase and pyruvate kinase evolved from a common ancestor.

Enolase or 2-phospho-D-glycerate hydrolase catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate, which in turn is converted by pyruvate kinase to pyruvate. We describe here the crystallographic determination of the structure of yeast enolase at high resolution (2.25 A) and an analysis of the structural homology between enolase, pyruvate kinase and triose phosphate isomerase. Each of the two subunits of enolase forms two distinctive domains. The larger domain (residues 143-420) is a regular 8-fold beta/alpha-barrel, as first found in triose phosphate isomerase, and later in pyruvate kinase and 11 other functionally different enzymes. An analysis of the molecular geometries of enolase and pyruvate kinase based on the roughly 8-fold symmetry of the barrel showed a structural homology better than expected for proteins related by convergent evolution. We argue that enolase and pyruvate kinase have evolved from a common ancestral multifunctional enzyme which could process phosphoenolpyruvate in both directions along the glycolytic pathway. There is structural and sequence evidence that muconate lactonizing enzyme later evolved from enolase.

Biological Evolution↗