Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.     

Residues 10-18 within the C5a receptor N terminus compose a binding domain for chemotaxis inhibitory protein of Staphylococcus aureus

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is excreted by the majority of S. aureus strains and is a potent inhibitor of C5a- and formylated peptide-mediated chemotaxis of neutrophils and monocytes. Recently, we reported that CHIPS binds to the C5a receptor (C5aR) and the formylated peptide receptor, thereby blocking activation by C5a and formylated peptides, respectively. The anaphylatoxin C5a plays an important role in host immunity and pathological inflammatory processes. For C5a a two-site binding model is proposed in which C5a initially binds the C5aR N terminus, followed by interaction of the C5a C-terminal tail with an effector domain on the receptor. We have shown here that CHIPS does not affect activation of the C5aR by a peptide mimic of the C5a C terminus. Moreover, CHIPS was found to bind human embryonic kidney 293 cells expressing only the C5aR N terminus. Deletion and mutation experiments within this C5aR N-terminal expression system revealed that the binding site of CHIPS is contained in a short stretch of 9 amino acids (amino acids 10-18), of which the aspartic acid residues at positions 10, 15, and 18 plus the glycine at position 12 are crucial. Binding studies with C5aR/C3aR and C5aR/IL8RA chimeras confirmed that CHIPS binds only to the C5aR N terminus without involvement of its extracellular loops. CHIPS may provide new strategies to block the C5aR, which may lead to the development of new C5aR antagonists.     

Two novel types of O-glycans on the mugwort pollen allergen Art v 1 and their role in antibody binding

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz (1)H and (13)C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core, which is substituted by a variable number (5-28) of alpha-arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) beta-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp(4) motifs in other hydroxyproline-rich glycoproteins, such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation, and/or digestion with alpha-arabinofuranosidase were used in enzyme-linked immunosorbent assay and immunoblot experiments with rabbit serum and with the sera of patients. Although we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked beta-arabinose residues appeared to react with the antibodies. Mono-beta-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive, carbohydrate determinant in plant proteins.     

Oligosaccharides implicated in recognition are predicted to have relatively ordered structures

Fucosylated O- and N-linked glycans are essential recognition molecules in plants and animals. To understand how they impart their functions, through interactions with proteins, requires a detailed analysis of structure and dynamics, but this is presently lacking. In this study, the three-dimensional structure and dynamics of three fucosylated oligosaccharides are investigated using a combination of high field (800 MHz) nuclear magnetic resonance and long (50 ns) molecular dynamics simulations in explicit water. Predictions from dynamics simulations were in agreement with nuclear Overhauser cross-peak intensities. Similarly, a theory of weak alignment in neutral media resulted in reasonable predictions of residual dipolar couplings for the trisaccharide fucosyllactose. However, for larger penta- and hexasaccharides (LNF-1 and LND-1), the anisotropic component of the alignment was underestimated, attributed to shape irregularities of the fucosyl branches on an otherwise linear core, being more pronounced in a singly branched than a doubly branched oligosaccharide. Simulations, confirmed by experiment, predicted fucosylated molecules that are restricted to librations about a single average conformation. This restriction is partly due to microscopic water interactions, which act to stabilize intramolecular hydrogen bonds and maintain tight and ordered conformations; a view not forthcoming from simpler, nonaqueous simulations. Such a conclusion is crucial for understanding how these molecules interact with proteins and impart their recognition properties.     

Crystal structure of the response regulator 02 receiver domain, the essential YycF two-component system of Streptococcus pneumoniae in both complexed and native states

A variety of bacterial cellular responses to environmental signals are mediated by two-component signal transduction systems comprising a membrane-associated histidine protein kinase and a cytoplasmic response regulator (RR), which interpret specific stimuli and produce a measured physiological response. In RR activation, transient phosphorylation of a highly conserved aspartic acid residue drives the conformation changes needed for full activation of the protein. Sequence homology reveals that RR02 from Streptococcus pneumoniae belongs to the OmpR subfamily of RRs. The structures of the receiver domains from four members of this family, DrrB and DrrD from Thermotoga maritima, PhoB from Escherichia coli, and PhoP from Bacillus subtilis, have been elucidated. These domains are globally very similar in that they are composed of a doubly wound alpha(5)beta(5); however, they differ remarkably in the fine detail of the beta4-alpha4 and alpha4 regions. The structures presented here reveal a further difference of the geometry in this region. RR02 is has been shown to be the essential RR in the gram-positive bacterium S. pneumoniae R. Lange, C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein, Gene 237:223-234, 1999; J. P. Throup, K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham, Mol. Microbiol. 35:566-576, 2000). RR02 functions as part of a phosphotransfer system that ultimately controls the levels of competence within the bacteria. Here we report the native structure of the receiver domain of RR02 from serotype 4 S. pneumoniae (as well as acetate- and phosphate-bound forms) at different pH levels. Two native structures at 2.3 A, phased by single-wavelength anomalous diffraction (xenon SAD), and 1.85 A and a third structure at pH 5.9 revealed the presence of a phosphate ion outside the active site. The fourth structure revealed the presence of an acetate molecule in the active site.     

Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton

CREC proteins constitute a family of EF-hand calcium binding proteins localized to the secretory pathway. Calumenin is the only member known to be secreted. Recently, it was shown that thrombin-activated thrombocytes liberate calumenin, which also is found in atherosclerotic lesions but not in normal vasculature. To study the possible effects of calumenin extracellularly, we used proteomic profiling of fibroblasts cultured in absence and in presence of calumenin. Using 2-DE and MS/MS, we show that normal fibroblasts contain several 28-29-kDa N-terminal and a 16-kDa C-terminal fragment of beta- or gamma-actin. Extracellularly added calumenin decreases the levels of both the N-terminal and C-terminal actin fragments, and, in addition, decreases the expression level of septin 2, which interacts with the actin cytoskeleton and is involved in cytokinesis. Labeling of S-phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin may have an autocrine or a paracrine effect on the cells in its vicinity, and, therefore, may be involved in the pathophysiology of thrombosis or in wound healing.     

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.     

Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substituents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual beta-(1-6)-glucan with (glycerol)2phosphate at the reducing end.     

Soil fauna communities and microbial respiration in high Arctic tundra soils at Zackenberg, Northeast Greenland

The soil fauna communities were described for three dominant vegetation types in a high arctic site at Zackenberg, Northeast Greenland. Soil samples were extracted to quantify the densities of mites, collembolans, enchytraeids, diptera larvae, nematodes and protozoa. Rates of microbial respiration were also assessed. Collembolans were found in highest densities in dry heath soil, about 130,000 individuals m⁻², more than twice as high as in mesic heath soils. Enchytraeids, diptera larvae and nematodes were also more abundant in the dry heath soil than in mesic heath soils, whereas protozoan densities (naked amoeba and heterotrophic flagellates) were equal. Respiration rate of unamended soil was similar in soil from the three plots. However, a higher respiration rate increase in carbon + nutrient amended soil and the higher densities of soil fauna (with the exception of mites and protozoa) in dry heath compared to the mesic heath soils indicated a higher decomposition rate here.     

Invasion of terrestrial enchytraeids into two postglacial tundras: North-eastern Greenland and the Arctic Archipelago of Canada (Enchytraeidae, Oligochaeta)

The aim was to study the invasion of terrestrial enchytraeids into the two postglacial tundras, North-eastern Greenland and the Arctic Archipelago of Canada. The majority of species recorded have a wide distribution in the Northern Hemisphere and the species diversity is considerably lower than that of potential source regions indicating either strong selection or varied dispersal ability. It appears that the Archipelago is influenced mainly from North America and North-eastern Greenland from Europe while the specialized fauna of the ancient Beringian tundra is of minor importance. The two alternative scenarios: (a) survival of a prepleistocene fauna in protected refugia within the area or (b) a postglacial re-invasion from outside are discussed, but the available data do not discriminate between these two possibilities. A total of 24 terrestrial enchytraeid taxa are recorded of which 22 are identified to the species level. Bryodrilus archipelagicus and Hemifridericia bivesiculata are described as new to science and Henlea groenlandica Černosvitov is revalidated. We hesitate to conclude that the new taxa described are endemic.