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Utrecht, Netherlands

Zapun A.,CNRS Institute of Pharmacology and Structural Biology | Zapun A.,French National Center for Scientific Research | Zapun A.,CEA Grenoble | Philippe J.,CNRS Institute of Pharmacology and Structural Biology | And 11 more authors.
ACS Chemical Biology | Year: 2013

Understanding the molecular basis of bacterial cell wall assembly is of paramount importance in addressing the threat of increasing antibiotic resistance worldwide. Streptococcus pneumoniae presents a particularly acute problem in this respect, as it is capable of rapid evolution by homologous recombination with related species. Resistant strains selected by treatment with β-lactams express variants of the target enzymes that do not recognize the drugs but retain their activity in cell wall building, despite the antibiotics being mimics of the natural substrate. Until now, the crucial transpeptidase activity that is inhibited by β-lactams was not amenable to in vitro investigation with enzymes from Gram-positive organisms, including streptococci, staphylococci, or enterococci pathogens. We report here for the first time the in vitro assembly of peptidoglycan using recombinant penicillin-binding proteins from pneumococcus and the precursor lipid II. The two required enzymatic activities, glycosyl transferase for elongating glycan chains and transpeptidase for cross-linking stem-peptides, were observed. Most importantly, the transpeptidase activity was dependent on the chemical nature of the stem-peptide. Amidation of the second residue glutamate into iso-glutamine by the recently discovered amido-transferase MurT/GatD is required for efficient cross-linking of the peptidoglycan. © 2013 American Chemical Society.


Briegel A.,California Institute of Technology | Briegel A.,Howard Hughes Medical Institute | Chen S.,California Institute of Technology | Koster A.J.,Institute of Biomembranes | And 4 more authors.
Methods in Enzymology | Year: 2010

Light and electron cryo-microscopy have each proven to be powerful tools to study biological structures in a near-native state. Light microscopy provides important localization information, while electron microscopy provides the resolution necessary to resolve fine structural details. Imaging the same sample by both light and electron cryo-microscopy is a powerful new approach that combines the strengths of both techniques to provide novel insights into cellular ultrastructure. In this chapter, the methods and instrumentation currently used to correlate light and electron cryo-microscopy are described in detail. © 2010 Elsevier Inc.


Ramadurai S.,Groningen Biomolecular science and Biotechnology Institute | Holt A.,Institute of Biomembranes | Schafer L.V.,University of Groningen | Krasnikov V.V.,Groningen Biomolecular science and Biotechnology Institute | And 4 more authors.
Biophysical Journal | Year: 2010

We investigated the effect of amino acid composition and hydrophobic length of α-helical transmembrane peptides and the role of electrostatic interactions on the lateral diffusion of the peptides in lipid membranes. Model peptides of varying length and composition, and either tryptophans or lysines as flanking residues, were synthesized. The peptides were labeled with the fluorescent label Alexa Fluor 488 and incorporated into phospholipid bilayers of different hydrophobic thickness and composition. Giant unilamellar vesicles were formed by electroformation, and the lateral diffusion of the transmembrane peptides (and lipids) was determined by fluorescence correlation spectroscopy. In addition, we performed coarsegrained molecular-dynamics simulations of single peptides of different hydrophobic lengths embedded in planar membranes of different thicknesses. Both the experimental and simulation results indicate that lateral diffusion is sensitive to membrane thickness between the peptides and surrounding lipids. We did not observe a difference in the lateral diffusion of the peptides with respect to the presence of tryptophans or lysines as flanking residues. The specific lipid headgroup composition of the membrane has a much less pronounced impact on the diffusion of the peptides than does the hydrophobic thickness. © 2010 by the Biophysical Society.


Bryde S.,Institute of Biomembranes | Bryde S.,French National Center for Scientific Research | Hennrich H.,Institute of Biomembranes | Verhulst P.M.,Institute of Biomembranes | And 4 more authors.
Journal of Biological Chemistry | Year: 2010

Members of the P4 subfamily of P-type ATPases catalyze phospholipid transport and create membrane lipid asymmetry in late secretory and endocytic compartments. P-type ATPases usually pump small cations and the transport mechanism involved appears conserved throughout the family. How this mechanism is adapted to flip phospholipids remains to be established. P 4-ATPases form heteromeric complexes with CDC50 proteins. Dissociation of the yeast P4-ATPase Drs2p from its binding partner Cdc50p disrupts catalytic activity (Lenoir, G., Williamson, P., Puts, C. F., and Holthuis, J. C. (2009) J. Biol. Chem. 284, 17956-17967), suggesting that CDC50 subunits play an intimate role in the mechanism of transport by P 4-ATPases. The human genome encodes 14 P4-ATPases while only three human CDC50 homologues have been identified. This implies that each human CDC50 protein interacts with multiple P4-ATPases or, alternatively, that some human P4-ATPases function without a CDC50 binding partner. Here we show that human CDC50 proteins each bind multiple class-1 P4-ATPases, and that in all cases examined, association with a CDC50 subunit is required for P4-ATPase export from the ER. Moreover, we find that phosphorylation of the catalytically important Asp residue in human P 4-ATPases ATP8B1 and ATP8B2 is critically dependent on their CDC50 subunit. These results indicate that CDC50 proteins are integral part of the P4-ATPase flippase machinery. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Puts C.F.,Institute of Biomembranes | Lenoir G.,Institute of Biomembranes | Lenoir G.,University Utrecht | Krijgsveld J.,Amherst College | And 3 more authors.
Journal of Proteome Research | Year: 2010

High-throughput analysis of protein-protein interactions can provide unprecedented insight into how cellular processes are integrated at the molecular level. Yet membrane proteins are often overlooked in these studies owing to their hydrophobic nature and low abundance. Here we used a proteomicsbased strategy with the specific intention of identifying membrane-associated protein complexes. One important aspect of our approach is the use of chemical cross-linking to capture transient and lowaffinity protein interactions that occur in living cells prior to cell lysis. We applied this method to identify binding partners of the yeast Golgi P4-ATPase Drs2p, a member of a conserved family of putative aminophospholipid transporters. Drs2p was endogeneously tagged with both a polyhistidine and a biotinylation peptide, allowing tandem-affinity purification of Drs2p-containing protein complexes under highly stringent conditions. Mass-spectrometric analysis of isolated complexes yielded one known and nine novel Drs2p binding partners. Binding specificity was verified by an orthogonal in vivo membrane protein interaction assay, confirming the efficacy of our method. Strikingly, three of the novel Drs2p interactors are involved in phosphoinositide metabolism. One of these, the phosphatidylinositol-4-phosphatase Sac1p, also displays genetic interactions with Drs2p. Together, these findings suggest that aminophospholipid transport and phosphoinositide metabolism are interconnected at the Golgi. © 2010 American Chemical Society.

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