Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin.

BACKGROUND: UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), catalyses the first committed step of bacterial cell wall biosynthesis and is a target for the antibiotic fosfomycin. The only other known enolpyruvyl transferase is 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme involved in the shikimic acid pathway and the target for the herbicide glyphosate. Inhibitors of enolpyruvyl transferases are of biotechnological interest as MurA and EPSP synthase are found exclusively in plants and microbes. RESULTS: The crystal structure of Escherichia coli MurA complexed with UDP-N-acetylglucosamine (UDP-GlcNAc) and fosfomycin has been determined at 1.8 A resolution. The structure consists of two domains with the active site located between them. The domains have a very similar secondary structure, and the overall protein architecture is similar to that of EPSP synthase. The fosfomycin molecule is covalently bound to the cysteine residue Cys115, whereas UDP-GlcNAc makes several hydrogen-bonding interactions with residues from both domains. CONCLUSIONS: The present structure reveals the mode of binding of the natural substrate UDP-GlcNAc and of the drug fosfomycin, and provides information on the residues involved in catalysis. These results should aid the design of inhibitors which would interfere with enzyme-catalyzed reactions in the early stage of the bacterial cell wall biosynthesis. Furthermore, the crystal structure of MurA provides a model for predicting active-site residues in EPSP synthase that may be involved in catalysis and substrate binding.

Crystal structure of the ffh and EF-G binding sites in the conserved domain IV of Escherichia coli 4.5S RNA.

BACKGROUND: Bacterial signal recognition particle (SRP), consisting of 4.5S RNA and Ffh protein, plays an essential role in targeting signal-peptide-containing proteins to the secretory apparatus in the cell membrane. The 4.5S RNA increases the affinity of Ffh for signal peptides and is essential for the interaction between SRP and its receptor, protein FtsY. The 4.5S RNA also interacts with elongation factor G (EF-G) in the ribosome and this interaction is required for efficient translation. RESULTS: We have determined by multiple anomalous dispersion (MAD) with Lu(3+) the 2.7 A crystal structure of a 4.5S RNA fragment containing binding sites for both Ffh and EF-G. This fragment consists of three helices connected by a symmetric and an asymmetric internal loop. In contrast to NMR-derived structures reported previously, the symmetric loop is entirely constituted by non-canonical base pairs. These pairs continuously stack and project unusual sets of hydrogen-bond donors and acceptors into the shallow minor groove. The structure can therefore be regarded as two double helical rods hinged by the asymmetric loop that protrudes from one strand. CONCLUSIONS: Based on our crystal structure and results of chemical protection experiments reported previously, we predicted that Ffh binds to the minor groove of the symmetric loop. An identical decanucleotide sequence is found in the EF-G binding sites of both 4.5S RNA and 23S rRNA. The decanucleotide structure in the 4.5S RNA and the ribosomal protein L11-RNA complex crystals suggests how 4.5S RNA and 23S rRNA might interact with EF-G and function in translating ribosomes.

Coenzyme binding in crystals of glyceraldehyde-3-phosphate dehydrogenase.

Apo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus and the partially saturated holo-enzyme can be crystallized isomorphously with the entire tetramer occupying the crystal asymmetric unit. For crystals that contain one molecule of NAD+ per tetramer the coenzyme is bound uniquely in one of the four available sites. The presence of NAD+ gives rise to nonequivalence in the binding of a heavy-atom compound to the subunits of the tetramer while for the apo-enzyme this binding is clearly symmetric. These results suggest that NAD binding gives rise to sequential ligand-induced structural changes of the tetramer, which may be responsible for the observed negative cooperativity in coenzyme binding.

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.

Coenzyme-induced conformational changes in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The structure of apo-glyceraldehyde-3-phosphate dehydrogenase (GAPDHase) from Bacillus stearothermophilus has been refined using a restrained least-squares method. The final crystallographic R-factor is 0.177 for all 53,315 reflections between 7.0 and 2.5 A. The resulting model has been analysed with respect to lattice interactions, molecular symmetry, temperature factors and solvent structure showing that, apart from local deviations due to intermolecular contact, the molecule exhibits a very high degree of local 222 symmetry. Analysis of differences between the structure of apo-GAPDHase and the previously refined holo-GAPDHase at 1.8 A resolution reveals details of conformational change in the enzyme induced by cofactor binding. The change, which was previously described as a rigid-body rotation of the coenzyme-binding domain with respect to the catalytic domain, is of more complex nature and involves relative shifts of several structural elements in the coenzyme-binding domain and some small changes in the catalytic domain. A possible mechanism of this conformational change is proposed based on the comparison of the refined structures and model-building studies. According to this mechanism, the adenosine moiety of NAD can initially bind to the protein in the apo-enzyme conformation. Several attractive interactions resulting from the initial binding of the coenzyme trigger conformational changes in the molecule of GAPDHase that: (1) create the productive nicotinamide-moiety binding site; (2) improve enzyme-coenzyme interactions at the adenosine moiety; (3) modify the active site to optimize the positioning of catalytic residues and ion-binding sites. Implications of the proposed mechanism for existing experimental data on binding of NAD analogues to GAPDHase are discussed.

Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution.

The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods. The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177. The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules. The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry. The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice. A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map. Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis. This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources. In the crystal both of the anion binding sites are occupied by sulphate ions. The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule.

Crystallization and preliminary X-ray analysis of porcine synovial collagenase.

Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group I4, with unit cell dimensions a = b = 160.0 A, c = 53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4 A.

Crystallization of inhibitor complexes of an N-terminal 24 kDa fragment of the DNA gyrase B protein.

A 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein has been crystallized in the presence of novobiocin. One crystal form has been obtained that is orthorhombic, P2(1)2(1)2(1), with unit cell dimensions a = 40.3 A, b = 47.7 A, c = 111.9 A. The asymmetric unit of this crystal form contains one molecule (Vm = 2.24 A3/Da). Complete native data have been collected to 2.5 A resolution. This same protein fragment has also been crystallized in the presence of GR122222X, an inhibitor that is structurally related to cyclothialidine. These crystals also exhibit P2(1)2(1)2(1) symmetry but have unit cell dimensions of a = 68.8 A, b = 68.6 A, c = 48.6 A. The Vm value of this crystal form is 2.39 A3/Da, assuming one molecule in the asymmetric unit, and native data have been collected to 2.0 A resolution. Molecular replacement studies of both complexes are underway.

Crystallization and preliminary X-ray analysis of Candida albicans phosphomannose isomerase.

Crystals of recombinant phosphomannose isomerase from Candida albicans have been obtained in a form suitable for X-ray diffraction analysis. The enzyme plays a key role in the biosynthesis of the mannan component of the fungal cell wall. It crystallizes in monoclinic space group C2, with cell dimensions a = 124.9 A, b = 52.9 A, c = 85.9 A and beta = 127.4 degrees. The crystals diffract to Bragg spacings beyond 1.7 A, native data have been collected to 2.4 A and a search for heavy-metal derivatives is in progress. The asymmetric unit contains one molecule of the enzyme (M(r) approximately 49,000) with a Vm of 2.3 A3/Da.

Crystallization and preliminary X-ray diffraction studies of human RANTES.

The chemotactic cytokine RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) is a potent chemoattractant and activator of a number of leukocytes, with a molecular mass of 8 kDa. Crystals of this protein have been grown from 100 mM sodium acetate buffer (pH 4.6) containing 200 mM magnesium acetate, with 20% (w/v) PEG 4000 and 6% (v/v) glycerol. The crystals grow as thick rods, which diffract to at least 1.8 A resolution on a rotating anode X-ray source. The crystals belong to space group p2(1)2(1)2(1) with unit cell dimensions a = 95.14 A, b = 57.58 A and c = 24.01 A with alpha = beta = gamma = 90 degrees. The asymmetric unit contains two molecules of the RANTES monomer, with a VM of 2.0 A(3)/Da.

Crystallization of the globular domain of histone H5.

The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed in Escherichia coli a gene that codes for the globular domain of H5. The protein produced in E. coli is functional in nucleosome binding assays. We have obtained crystals of the protein that diffract to beyond 2.5 A (1 A = 0.1 nm) resolution. The crystals are orthorhombic with unit cell dimensions of a = 80.1 A, b = 67.5 A and c = 38.0 A.

X-ray and spectrophotometric studies of the binding of proflavin to the S1 specificity pocket of human alpha-thrombin.

Proflavin can be used to study the interactions of inhibitors and substrates with thrombin by monitoring the changes in the visible absorption spectrum that occur on dye displacement. We have used microspectrophotometric methods to investigate the binding of proflavin to crystals of an alpha-thrombin-hirugen complex and have determined the structure by X-ray crystallography. The proflavin molecule binds in the S1 pocket of the enzyme with one of the amino groups hydrogen bonded to the carboxylate of Asp-189 while the protonated ring nitrogen is hydrogen bonded to the carbonyl of Gly-219. This result indicates that the proflavin displacement assay can be used to specifically monitor the binding of inhibitors to the S1 pocket.

The nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis.

Directed mutagenesis has been used to study the nicotinamide subsite of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Residue Asn313 is involved together with the carboxyamide moiety of the nicotinamide ring in a complex network of hydrogen bonding interactions which fix the position of the pyridinium ring of NAD to which hydride transfer occurs at the C-4 position in the catalytic reaction. The asparagine side-chain has been replaced by that of the Thr and Ala residues and results in mutants with very similar properties. Both mutants show much weaker binding of NAD and lower catalytic efficiency. The mutant Asn313----Thr still exhibits strict B-stereospecificity in hydride transfer and retains the property of negative co-operativity in NAD binding. These experiments strongly suggest that the mutant enzyme undergoes the apo----holo sub-unit structural transition associated with coenzyme binding but that the nicotinamide ring is no longer as rigidly held in its pocket as in the wild type enzyme. The results shed light on the details of the molecular interactions which are responsible for negative co-operativity in this enzyme.

A series of penicillin-derived C2-symmetric inhibitors of HIV-1 proteinase: structural and modeling studies.

The binding modes of a series of penicillin-derived C2 symmetric dimer inhibitors of HIV-1 proteinase were investigated by NMR, protein crystallography, and molecular modeling. The compounds were found to bind in a symmetrical fashion, tracing and S-shaped course through the active site, with good hydrophobic interactions in the S1/S1' and S2/S2' pockets and hydrogen bonding of inhibitor amide groups. Interactions with the catalytic aspartates appeared poor and the protein conformation was very similar to that seen in complexes with peptidomimetics, in spite of the major differences in ligand structure.

Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from influenza virus types A and B.

The first paper in this series (see previous article) described structure-activity studies of carboxamide analogues of zanamivir binding to influenza virus sialidase types A and B and showed that inhibitory activity of these compounds was much greater against influenza A enzyme. To understand the large differences in affinities, a number of protein-ligand complexes have been investigated using crystallography and molecular dynamics. The crystallographic studies show that the binding of ligands containing tertiary amide groups is accompanied by the formation of an intramolecular planar salt bridge between two amino acid residues in the active site of the enzyme. It is proposed that the unexpected strong binding of these inhibitors is a result of the burial of hydrophobic surface area and salt-bridge formation in an environment of low dielectric. In sialidase from type A virus, binding of the carboxamide moeity and salt-bridge formation have only a minor effect on the positions of the surrounding residues, whereas in type B enzyme, significant distortion of the protein is observed. The results suggest that the decreased affinity in enzyme from influenza B is directly correlated with the small changes that occur in the amino acid residue interactions accompanying ligand binding. Molecular dynamics calculations have shown that the tendency for salt-bridge formation is greater in influenza A sialidase than influenza B sialidase and that this tendency is a useful descriptor for the prediction of inhibitor potency.

Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification.

The active site of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains two anion recognition sites which have been attributed to the phosphate binding of the substrates, namely, glyceraldehyde 3-phosphate (Ps site) and inorganic phosphate (Pi site) [Moras et al. (1975) J. Biol. Chem. 250, 9137-9162]. In order to probe the role of both sites during the catalytic event, Arg 195 from the Pi site and Arg 231 from the Ps site of the Bacillus stearothermophilus enzyme have been changed to Leu and Gly, respectively, by site-directed mutagenesis. A comparative study of the chemical reactivity of the mutants and wild type toward 2,3-butanedione revealed a similarly high reactivity only for the R195L mutant and wild type, suggesting that only Arg 231 is chemically reactive toward 2,3-butanedione and that its reactivity is not influenced by the presence of the residue Arg 195, which is only 4 A distant. The kinetic consequences of the mutations were also analyzed for the consecutive steps in the forward catalytic reaction. The replacement of Arg 195 by Leu leads to a marked decrease of the rate of the first steps of the reaction which lead to the acylenzyme formation, in particular, the rate of enzyme-substrate association, while these steps occur at a similar or higher rate when Arg 231 is replaced by Gly. Furthermore, the mutations R195L and R231G also result in a 550-fold and 16,400-fold decrease in the second-order rate constant of phosphorolysis. This step becomes rate-determining for the R195L mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148.

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase, Ser148----Ala, was produced by oligonucleotide-directed mutagenesis. The study of the catalytic properties of this mutant has shown that this mutation significantly affects the Michaelis constant of inorganic phosphate and to a lesser extent that of 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate. This result is consistent with model-building studies which show that, for the phosphorylation step of catalysis, inorganic phosphate must bind to the anion recognition site designated Pi with the C(3) phosphate of the acyl-enzyme intermediate in the alternative anion site Ps. Studies of the enantiomeric specificity using D- and L-glyceraldehyde as substrates show that the hydroxyl group of Ser148, combined with the presence of the C(3) phosphate of the substrate, enhances stereospecificity as well as catalysis. However, the stereospecific effect cannot be a consequence of the direct interaction of Ser148 with the C(2)-hydroxyl of the substrate. The changed Km for glyceraldehyde-3-phosphate suggests that the initial step of hemithioacetal formation may take place with its C(3) phosphate bound in the Pi site. This supports the molecular mechanism proposed by Moody (1984). Therefore, catalysis could be enhanced through interactions of the serine hydroxyl group not only with inorganic phosphate but also with the C(3) phosphate of glyceraldehyde-3-phosphate.