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1.  Ovalbumin-related Protein X Is a Heparin-binding Ov-Serpin Exhibiting Antimicrobial Activities* 
The Journal of Biological Chemistry  2013;288(24):17285-17295.
Background: Ovalbumin-related protein X (OVAX) is an uncharacterized ovalbumin-serpin.
Results: This egg white-specific serpin lacks protease inhibitory activity, but unlike its ovalbumin homolog, OVAX exhibits antibacterial properties, partly through its heparin-binding site(s).
Conclusion: OVAX, a non-inhibitory serpin is a heparin-binding molecule with antibacterial activity.
Significance: OVAX participates in egg defense and constitutes a natural agent against Listeria and Salmonella.
Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys367-His368 is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.
doi:10.1074/jbc.M113.469759
PMCID: PMC3682532  PMID: 23615912
Glycoprotein; Heparin Binding Protein; Innate Immunity; Protein Structure; Serpin; Antibacterial Proteins; Chicken Egg
2.  Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium 
The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the −10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with PramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (KD [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted PramA of an MDR S. Typhimurium clinical isolate than for the wild-type PramA (KD = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
doi:10.1128/AAC.05444-11
PMCID: PMC3264254  PMID: 22123696
3.  Crystal Structure of Diedel, a Marker of the Immune Response of Drosophila melanogaster 
PLoS ONE  2012;7(3):e33416.
Background
The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines.
Methodology/Principal Findings
We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 Å resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel β-sheet covered by an α-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families.
Conclusion/Significance
Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small family of proteins present in drosophilids, aphids and DNA viruses infecting lepidopterans. Diedel is an extracellular protein composed of two sub-domains. Two special structural features (hydrophobic surface patch and cis/trans conformation for proline 52) may indicate a putative interaction site, and support an extra cellular signaling function for Diedel, which is in accordance with its proposed role as negative regulator of the JAK/STAT signaling pathway.
doi:10.1371/journal.pone.0033416
PMCID: PMC3307722  PMID: 22442689
4.  Expression, purification, crystallization and preliminary X-ray analysis of the N-terminal domain of GNBP3 from Drosophila melanogaster  
Crystals of the N-terminal domain of Gram-negative bacteria-binding protein 3 of D. melanogaster grown from PEG solutions are monoclinic (space group C2) and diffract to 1.7 Å resolution.
Gram-negative bacteria-binding protein 3 (GNBP3) is a pattern-recognition receptor which contributes to the defensive response against fungal infection in Drosophila. The protein consists of an N-terminal domain, which is considered to recognize β-glucans from the fungal cell wall, and a C-terminal domain, which is homologous to bacterial glucanases but devoid of activity. The N-terminal domain of GNBP3 (GNBP3-Nter) was successfully purified after expression in Drosophila S2 cells. Diffraction-quality crystals were produced by the hanging-drop vapour-diffusion method using PEG 2000 and PEG 8000 as precipitants. Preliminary X-ray diffraction analysis revealed that the GNBP3-Nter crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 134.79, b = 30.55, c = 51.73 Å, β = 107.4°, and diffracted to 1.7 Å using synchrotron radiation. The asymmetric unit is expected to contain two copies of GNBP3-Nter. Heavy-atom derivative data were collected and a samarium derivative showed one high-occupancy site per molecule.
doi:10.1107/S1744309109014997
PMCID: PMC2795588  PMID: 19724120
GNBP3; N-terminal domain; Drosophila melanogaster; pattern-recognition receptors
5.  5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases 
Nucleic Acids Research  2011;39(14):6277-6290.
DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.
doi:10.1093/nar/gkr215
PMCID: PMC3152353  PMID: 21486746

Results 1-5 (5)