Synthetic chemistry. Sugaring vancomycin.
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Carbohydrates↗
Biomedical subjects
D H Williams
Publications and source records attributed to D H Williams.
Structure of the protein tyrosine kinase domain of C-terminal Src kinase (CSK) in complex with staurosporine.
The crystal structure of the kinase domain of C-terminal Src kinase (CSK) has been determined by molecular replacement, co-complexed with the protein kinase inhibitor staurosporine (crystals belong to the space group P21212 with a=44.5 A, b=120.6 A, c=48.3 A). The final model of CSK has been refined to an R-factor of 19.9 % (Rfree=28.7 %) at 2.4 A resolution. The structure consists of a small, N-terminal lobe made up mostly of a beta-sheet, and a larger C-terminal lobe made up mostly of alpha-helices. The structure reveals atomic details of interactions with staurosporine, which binds in a deep cleft between the lobes. The polypeptide chain fold of CSK is most similar to c-Src, Hck and fibroblast growth factor receptor 1 kinase (FGFR1K) and most dissimilar to insulin receptor kinase (IRK). Interactions between the N and C-terminal lobe are mediated by the bound staurosporine molecule and by hydrogen bonds. In addition, there are several water molecules forming lobe-bridging hydrogen bonds, which may be important for maintaining the catalytic integrity of the kinase. Furthermore, the conserved Lys328 and Glu267 residues utilise water in the formation of a molecular pivot which is essential in allowing relative movement of the N and C-terminal lobes. An analysis of the residues around the ATP-binding site reveals structural differences with other protein tyrosine kinases. Most notable of these are different orientations of the conserved residues Asp332 and Phe333, suggesting that inhibitor binding proceeds via an induced fit. These structural observations have implications for understanding protein tyrosine kinase catalytic mechanisms and for the design of ATP-mimicking inhibitors of protein kinases.
Amino Acid Sequence↗
Human MAFA has alternatively spliced variants.
Human mast cell function-associated antigen (MAFA) cDNA has been cloned. This molecule is similar to the rat form having an intracellular domain containing a putative immunoreceptor tyrosine inhibition motif and an extracellular C type lectin-like domain. However, in contrast to rat MAFA, the amino acid sequence suggests the presence of two additional extracellular N-linked glycosylation sites. In addition, alternative mRNA transcripts are observed that differ substantially from those found in the rat.
Alternative Splicing↗
Surface plasmon resonance analysis at a supported lipid monolayer.
Methods for the formation of supported lipid monolayers on top of a hydrophobic self assembled monolayer in a surface plasmon resonance instrument are described. Small unilamellar vesicles absorb spontaneously to the surface of the hydrophobic self-assembled monolayer to form a surface which resembles the surface of a cellular membrane. Lipophilic ligands, such as small acylated peptides or glycosylphosphatidylinositol-anchored proteins, were inserted into the absorbed lipid and binding of analytes to these ligands was analysed by surface plasmon resonance. Conditions for the formation of lipid monolayers have been optimised with respect to lipid type, chemical and buffer compatibility, ligand stability and reproducibility.
Anti-Bacterial Agents↗
Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment.
The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.
Animals↗
Ro 09-2210 exhibits potent anti-proliferative effects on activated T cells by selectively blocking MKK activity.
By using high throughput screening of microbial broths, we have identified a compound, designated Ro 09-2210, which is able to block anti-CD3 induced peripheral blood T cell activation with an IC50 = 40 nM. Ro 09-2210 was also able to block antigen-induced IL-2 secretion with an IC50 = 30 nM, but was considerably less potent at blocking Ca2+ flux stimulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cells using a variety of reporter gene constructs and showed effective inhibition of phorbol ester/ionomycin-induced NF-AT activation and anti-CD3 induced NF-AT with IC50 = 7.7 and 10 nM, respectively. Ro 09-2210 was also able to inhibit phorbol ester/ionomycin-induced activation of AP1 with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter constructs (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 activation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibiting an activator of AP-1 which was upstream of c-jun and downstream of ras signaling. To investigate further, we then purified a number of different kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), and showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (IC50 = 59 nM).
DNA-Binding Proteins↗
The influence of selected herd factors and a badger-intervention tuberculosis-control programme on the risk of a herd-level trade restriction to a bovine population in Ireland.
The risk of a trade restriction to cattle herds between 1990-1994, as a result of failing the tuberculin skin test or disclosure of visible lesions in the abattoir, was compared in two areas in East Offaly, Ireland. Cattle herds from the Project area where badgers were systematically removed over a 7-year period (1989-1995) were compared to herds from a Control area where no wildlife-intervention programme had taken place. A Cox proportional hazard model indicated, for example, that the risk of a herd-level trade restriction to herds with 30 animals in the Control area was 1.4 times the risk to similar-sized herds in the Project area. The study provides evidence that a badger-control programme is effective in reducing the risk of a trade restriction to cattle herds as the badger represents an important reservoir of Mycobacterium bovis infection for cattle.
Animals↗
An analysis of the origins of a cooperative binding energy of dimerization.
The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.
Anti-Bacterial Agents↗
The effect of a badger removal programme on the incidence of tuberculosis in an Irish cattle population.
The risk of a confirmed tuberculous herd restriction was examined using a logistic model for herds involved in the East Offaly Badger Research Project, Ireland, from 1988-1995. Cattle herds present in the badger-removal area had a significantly lower proportion of new confirmed tuberculous herd restrictions compared with cattle from an area where no systematic badger removal was attempted.
Animals↗
Modelling the prevalence of Echinococcus and Taenia species in small ruminants of different ages in northern Jordan.
A base-line survey of Echinococcus granulosus, Taenia hydatigena and T ovis were undertaken in order to investigate the transmission dynamics of these parasites in northern Jordan. Intensity of E. granulosus infection, in sheep, increased with age in a linear fashion whilst the asymptotic prevalence was one. This implied that E. granulosus is in an endemic steady state with no evidence of protective immunity in the intermediate host. The mean number of cysts increased by 1.66 per year with approximately 0.320 infections per year, each infection consisting of 598 eggs to produce 5.2 cysts. The basic reproduction ratio (R0) was estimated to be 1.5-1.8. A similar pattern was suggested with E. granulosus in goats but the infection pressure appeared to be lower with only 0.128 cysts per year. Although infection in goats appeared to be endemic there was some evidence of departure from the model which might indicate that the model needs adjusting for this species. In the case of T. hydatigena the host age-intensity helminth distribution indicated that this parasite was hyperendemic in both sheep and goats, implying regulation by intermediate host immunity. Consequently, R0 was determined from asymptotic prevalence curves for T hydatigena and was calculated to be 4.0 and 3.1 in sheep and goats, respectively. The lower R0 in goats, together with the higher asymptotic age-intensity and age-prevalences, indicates that goats acquire immunity more slowly to T hydatigena in comparison to sheep. Taenia ovis was not detected in any animals.
Animals↗
Sequencing and analysis of genes involved in the biosynthesis of a vancomycin group antibiotic.
BACKGROUND: The emergence of resistance to vancomycin, the drug of choice against methicillin-resistant Staphylococcus aureus, in enterococci has increased the need for new antibiotics. As chemical modification of the antibiotic structure is not trivial, we have initiated studies towards enzymatic modification by sequencing the DNA coding for the biosynthesis of chloroeremomycin (also known as A82846B and LY264826). RESULTS: Analysis of 72 kilobases of genomic DNA from Amycolatopsis orientalis, the organism that produces chloroeremomycin, revealed the presence of 39 putative genes, including those coding for the biosynthesis of the antibiotic. Translation and subsequent comparison with known proteins in public databases identified enzymes responsible for the biosynthesis of the heptapeptide backbone and 4-epi-vancosamine, as well as those for chlorination and oxidation reactions involved in the biosynthesis of chloroeremomycin. CONCLUSIONS: The genes responsible for the biosynthesis of chloroeremomycin have been identified, and selective expression of these genes could lead to the synthesis of new potent glycopeptide antibiotics.
Actinobacteria↗
19F NMR in the measurement of binding affinities of chloroeremomycin to model bacterial cell-wall surfaces that mimic VanA and VanB resistance.
BACKGROUND: The emergence of bacteria that are resistant to vancomycin, the drug of choice against methicillin-resistant Staphylococcus aureus, has made the study of the binding characteristics of glycopeptides to biologically relevant depsipeptides important. These depsipeptides, terminating in D-alanyl-D-lactate, mimic the cell-wall precursors of resistant bacteria. RESULTS: The use of 19F-labelled ligands in the study of the therapeutically important vancomycin series of antibiotics is demonstrated. The substantial simplification of spectra that occurs when such labelled ligands are employed is used in the measurement of binding affinities of depsipeptides to chloroeremomycin (CE). Large enhancements of binding affinities are found at a model bacterial cell-wall surface (constituted from depsipeptides that are anchored into vesicles) relative to those measured in free solution. CONCLUSIONS: Surface-enhanced binding, previously shown for strongly dimerizing glycopeptide antibiotics to normal -D-alanyl-D-alanine-terminating cell-wall precursors, is now demonstrated for CE to the surface of models of VanA- and VanB-resistant bacteria. The effect of depsipeptide chain length is shown to be critically important in producing and maximizing this enhancement.
Anti-Bacterial Agents↗
Cleavage of rhamnose from ristocetin A removes its ability to induce platelet aggregation.
Ristocetin A is used clinically as a tool for the measurement of von Willebrand protein due to its ability to induce the aggregation of platelets in plasma being proportional to the concentration of von Willebrand protein. The enzymatically induced cleavage of alpha-L-rhamnose from the tetrasaccharide of ristocetin A removes this ability to induce platelet aggregation in plasma, providing a possible insight into the structural basis for this effect.
Carbohydrate Conformation↗
Induction of asymmetry into homodimers.
The self-regulation of biological signalling receptors via homodimerization is discussed in relation to the symmetry changes occurring when these receptors bind their target ligand. The idea of positive and negative cooperativity between dimerization and ligand binding, mediated by changes in the symmetry of the system as a source of signalling control is considered; an analogy made with the homodimerization of a glycopeptide antibiotic, ristocetin A, which displays negative cooperativity. Finally, the regulation of the bacterial aspartate receptor and the human growth hormone receptor is discussed as a function of ligand-induced asymmetry.
Animals↗
A limitation of two-state analysis for transitions between disordered and weakly ordered states.
BACKGROUND: The stability of the secondary structure of particular peptide regions is often used to investigate the involvement of the region in protein folding. When analysing the relatively small populations of associated states that are formed by weak interactions (i.e. those interactions that are comparable to thermal energies), it is common practice to characterise the associated state by a parameter that is measured when this state is highly occupied. The accuracy of this method, however, has not yet been determined. RESULTS: Using as a model the vancomycin group of antibiotics, either forming dimers or binding to cell wall precursors, we have investigated the dependence of the limiting (i.e. fully associated) chemical shifts of two protons on the equilibrium constants for the formation of the fully associated states. The chemical shift shows a large variation with the equilibrium constant for the formation of the fully associated state. CONCLUSIONS: The results demonstrate, in two systems, that a parameter representing a fully associated state (chemical shift) varies greatly with the equilibrium constant for the formation of that associated state. The results have implications for two-state analyses of populations of protein fragments in which a parameter representing the fully associated state is taken to be independent of the equilibrium constant for its formation. Using two-state analysis to determine the population of associated states of protein fragments could result in an underestimation of the population of these associated states.
Magnetic Resonance Spectroscopy↗
Semiquantitation of cooperativity in binding of vancomycin-group antibiotics to vancomycin-susceptible and -resistant organisms.
The association of vancomycin group antibiotics with the growing bacterial cell wall was investigated by using the cell wall precursor analog di-N-acetyl-Lys-D-Ala-D-Ala in competition binding experiments. The affinities of the antibiotics for the -D-Ala-D-Ala-containing cell wall precursors of Bacillus subtilis ATCC 6633 (a model for vancomycin-susceptible gram-positive bacteria) and for the -D-Ala-D-Lac-containing cell wall precursors of Leuconostoc mesenteroides (a model for vancomycin-resistant strains of Enterococcus faecium and Enterococcus faecalis) were determined by a whole-cell assay. The binding of strongly dimerizing antibiotics such as eremomycin to the bacterial surface was thus shown to be enhanced by up to 2 orders of magnitude (relative to the binding in free solution) by the chelate effect, whereas weakly dimerizing antibiotics like vancomycin and antibiotics carrying lipid tails (teicoplanin) benefited less (ca. 1 order of magnitude). The affinity measured in this way correlates well with the MIC of the antibiotic, and a consequence of this is that future design of semisynthetic vancomycin-group antibiotics should attempt to incorporate chelate effect-enhancing structural features.
Anti-Bacterial Agents↗
Structural elucidation of XR586, a peptaibol-like antibiotic from Acremonium persicinum.
A novel peptide, XR586, has been isolated from fermentations of Acremonium persicinum (Xenova culture collection number X21488). The structure of XR586 has been elucidated by means of NMR spectroscopy, electrospray and fast-atom bombardment MS, derivatization and enzymic digestion. It has been shown to be helical by CD measurements. XR586 shows many structural and conformational features in common with peptaibols, particularly the zervamicins. Peptaibol antibiotics are peptides, typically of 15-20 residues, containing a large proportion of alpha-aminoisobutyric acid (Aib) residues. These peptides adopt a helical conformation in solution and display anti-bacterial and toxic properties due to their ability to form pores in membranes. However, while XR586 contains several Aib residues, it lacks a terminal phenylalaninol and terminates in the sequence Phe-Gly. The lack of reduction of the penultimate residue at the C-terminus may indicate that this step is normally at the end of the biosynthetic pathway of peptaibols and occurs with cleavage of Gly. The 1H chemical shift assignments of XR586 are reported in Supplementary Publication SUP 50179 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1996) 313, 9 ("Deposition of data').
Acremonium↗
Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.
Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.
Adaptor Proteins, Signal Transducing↗