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31.
Characterization of protein-protein interactions that are critical to the specific function of many biological systems has become a primary goal of structural biology research. Analysis of these interactions by structural techniques is, however, challenging due to inherent limitations of the techniques and because many of the interactions are transient, and suitable complexes are difficult to isolate. In particular, structural studies of large protein complexes by traditional solution NMR methods are difficult due to a priori requirement of extensive assignments and a large number of intermolecular restraints for the complex. An approach overcoming some of these challenges by utilizing orientational restraints from residual dipolar couplings collected on solution NMR samples is presented. The approach exploits existing structures of individual components, including the symmetry properties of some of these structures, to assemble rapidly models for relatively large protein-protein complexes. An application is illustrated with a 95 kDa homotrimeric complex of the acyltransferase protein, LpxA (UDP-N-acetylglucosamine acyltransferase), and acyl carrier protein. LpxA catalyzes the first step in the biosynthesis of the lipid A component of lipopolysaccharide in Gram-negative bacteria. The structural model generated for this complex can be useful in the design of new anti-bacterial agents that inhibit the biosynthesis of lipid A. 相似文献
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Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4'-kinase, is present in Francisella, no 4'-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4'-phosphate residue from tetra- and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4'-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4'-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4'-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4'-position. Expression of LpxF in wild-type E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria. 相似文献
34.
Raetz CR Garrett TA Reynolds CM Shaw WA Moore JD Smith DC Ribeiro AA Murphy RC Ulevitch RJ Fearns C Reichart D Glass CK Benner C Subramaniam S Harkewicz R Bowers-Gentry RC Buczynski MW Cooper JA Deems RA Dennis EA 《Journal of lipid research》2006,47(5):1097-1111
The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2. 相似文献
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36.
Nathanael J. Spann Lana X. Garmire Jeffrey G. McDonaldDavid S. Myers Stephen B. MilneNorihito Shibata Donna ReichartJesse N. Fox Iftach ShakedDaniel Heudobler Christian R.H. Raetz Elaine W. WangSamuel L. Kelly M. Cameron SullardsRobert C. Murphy Alfred H. Merrill Jr.H. Alex Brown Edward A. DennisAndrew C. Li Klaus LeySotirios Tsimikas Eoin FahyShankar Subramaniam Oswald QuehenbergerDavid W. Russell Christopher K. Glass 《Cell》2012,151(1):138-152
37.
Escherichia coli lipid A is a hexaacylated disaccharide of glucosamine with secondary laurate and myristate chains on the distal unit. Hexaacylated lipid A is a potent agonist of human Toll-like receptor 4, whereas its tetra- and pentaacylated precursors are antagonists. The inner membrane enzyme LpxL transfers laurate from lauroyl-acyl carrier protein to the 2'- R-3-hydroxymyristate moiety of the tetraacylated lipid A precursor Kdo 2-lipid IV A. LpxL has now been overexpressed, solubilized with n-dodecyl beta- d-maltopyranoside (DDM), and purified to homogeneity. LpxL migration on a gel filtration column is consistent with a molecular mass of 80 kDa, suggestive of an LpxL monomer (36 kDa) embedded in a DDM micelle. Mass spectrometry showed that deformylated LpxL was the predominant species, noncovalently bound to as many as 12 DDM molecules. Purified LpxL catalyzed not only the formation in vitro of Kdo 2-(lauroyl)-lipid IV A but also a slow second acylation, generating Kdo 2-(dilauroyl)-lipid IV A. Consistent with the Kdo dependence of crude LpxL in membranes, Kdo 2-lipid IV A is preferred 6000-fold over lipid IV A by the pure enzyme. Sequence comparisons suggest that LpxL shares distant homology with the glycerol-3-phosphate acyltransferase (GPAT) family, including a putative catalytic dyad located in a conserved H(X) 4D/E motif. Mutation of H132 or E137 to alanine reduces specific activity by over 3 orders of magnitude. Like many GPATs, LpxL can also utilize acyl-CoA as an alternative acyl donor, albeit at a slower rate. Our results show that the acyltransferases that generate the secondary acyl chains of lipid A are members of the GPAT family and set the stage for structural studies. 相似文献
38.
The biosynthesis of gram-negative endotoxin. A novel kinase in Escherichia coli membranes that incorporates the 4'-phosphate of lipid A 总被引:7,自引:0,他引:7
Extracts of Escherichia coli contain an enzyme that generates the beta,1----6 linkage of lipid A from fatty-acylated monosaccharide precursors, according to the reaction: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2,3-diacyl-GlcN (beta, 1----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B. L., Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859). We now describe a membrane-bound kinase that phosphorylates the 4'-position of the above tetraacyldisaccharide 1-phosphate product. The lipid A 4'-kinase is distinct from the diglyceride kinase of E. coli. When crude membrane preparations are employed, several nucleoside triphosphates are able to support the phosphorylation of the tetraacyldisaccharide 1-phosphate, but ATP is the most efficient. The 4'-kinase requires Mg2+ and is stimulated by phospholipids, especially cardiolipin. Under optimal conditions the specific activity in crude extracts is 0.5 nmol/min/mg. The enzyme is rapidly inactivated by preincubation in the presence of detergents, such as Nonidet P-40 or octylglucoside, but phosphoenolpyruvate and glycerol stabilize the enzyme. The product generated in vitro has been characterized by fast atom bombardment mass spectrometry and by 1H and 31P NMR spectroscopy. Those analyses confirm that the 4' hydroxyl is the site of phosphorylation. The 4'-kinase reported here is likely to represent a key step in the de novo biosynthesis of lipid A. 相似文献
39.
CR Taylor 《Biotechnic & histochemistry》2014,89(6):419-423
The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides. 相似文献
40.
Two-thirds of the lipid A in wild-type Escherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'. The remaining lipid A contains a monophosphate substituent at position 4' and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 degrees C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-L-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives. 相似文献