X-ray absorption spectroscopy of a new zinc site in the fur protein from Escherichia coli.

International audienceThe zinc K-edge X-ray absorption spectra of the Fur (ferric uptake regulation) protein isolated from Escherichia coli have been analyzed in frozen solution to determine details of the zinc coordination. The spectra of apoFur and of the cobalt-substituted protein have been analyzed and compared in order to see the influence of the cobalt incorporation on the geometry of the zinc site. EXAFS analysis gave for both samples (apoFur and CoFur) a tetrahedral environment for the zinc atom with two sulfur donor ligands at a distance of 2.3 A from the zinc and two N/O donor ligands at 2.0 A. The two sulfur donor ligands are probably two of the four cysteines present in each Fur monomer and could be Cys92 and Cys95, which are known from mutagenesis studies to be essential for Fur activity [Coy, M., Doyle, C., Besser, J., and Neilands, J. B. (1994) BioMetals 7, 292-298]. The distances obtained from our fits were always too short to be compatible with penta or hexa coordination. The typical pattern observed for the Fourier transform of the EXAFS oscillations suggests the presence of at least one imidazole ligand. The XANES of these two forms of the protein are similar but significantly different. This suggests a change of the conformation of the zinc site upon cobalt incorporation. The present study provides the first unambiguous evidence for the presence of a structural zinc site in the Fur protein from Escherichia coli

Electrospray ionization mass spectrometry analysis of the apo- and metal-substituted forms of the Fur protein.

International audienceFur has been purified and reconstituted with Co2+ and Mn2+. The ESI-MS spectra of the apoprotein as well as Mn-Fur and Co-Fur under acidic denaturating conditions showed the existence of two species of molecular mass 16,660 +/- 3 and 16,792 +/- 3 Da, which correspond, respectively, to the N-terminal methionine 'excised' or 'non-excised' forms of the monomer. This result proves the absence of any other post-translational modification or modification due to metal incorporation. On the other hand, under soft conditions, ESI spectra provided for the first time direct evidence for dimeric metal-containing forms in solution

Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli: chemical modification and mass spectrometry analysis.

International audienceSelective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein. The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry. Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation by the zinc atom. Enzymatic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 and Cys95, were thus assigned as zinc ligands. Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site. Furthermore, Cys132 was shown to be the fastest reacting cysteine, implying it is a surface-exposed residue

Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

International audienceAbstract G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin

SIN3 acts in distinct complexes to regulate the germline transcriptional program in C. elegans

Abstract The SIN3 transcriptional coregulator influences gene expression through multiple interactions that include histone deacetylases (HDACs). Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability (ID)/autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here we show that loss of SIN-3, the single SIN3 homologue in Caenorhabditis elegans , results in maternal effect sterility associated with deregulation of the germline transcriptome, including desilencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific HDACs, and show that they differentially contribute to fertility. Single cell smFISH reveals that in sin-3 mutants, the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN3. Together, this work provides a powerful framework for the in vivo study of SIN3 and associated proteins

Spectroscopic and saturation magnetization properties of the manganese- and cobalt-substituted Fur (ferric uptake regulation) protein from Escherichia coli.

International audienceThe Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment

Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology

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