Miquel Pons

Publications (116) View all

• Article: Protein oligomers studied by solid-state NMR: the case of full-length nucleoid associated protein H-NS.

  Marie Renault, Jesús García, Tiago N Cordeiro, Marc Baldus, Miquel Pons
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  ABSTRACT: Members of the histone-like nucleoid structuring protein (H-NS) family play roles both as architectural proteins and as modulators of gene expression in Gram-negative bacteria. The H-NS protein participates in modulatory processes that respond to environmental changes in osmolarity, pH or temperature. H-NS oligomerization is essential for its activity. Structural models from different truncated forms are available. However, high-resolution structural details of full-length protein H-NS and its DNA bound state have largely remained elusive. We report on progress to characterize the biologically active H-NS oligomers using solid-state NMR spectroscopy (ssNMR). We have compared uniformly ((13) C,(15) N)-labeled ssNMR preparations of the isolated N-terminal region (H-NS 1-47) and full-length H-NS (H-NS 1-137). In both cases, we obtained ssNMR spectra of good quality and characteristic of well-folded proteins. Analysis of two- and three-dimensional ((13) C,(13) C) and ((15) N,(13) C) correlation experiments conducted at high field led to assignments of residues located in different topological regions of the free full-length H-NS protein. These findings confirm that the structure of the (N-terminal) dimerization domain is conserved in the oligomeric full-length protein. Small changes in the dimerization interface suggested by localized chemical shift variations between solution and solid-state spectra may be relevant for DNA recoginition. This article is protected by copyright. All rights reserved.
  FEBS Journal 04/2013; · 3.79 Impact Factor
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  Article: On the origin of the selectivity of plasmidic H-NS towards horizontally acquired DNA. Linking H-NS oligomerization and cooperative DNA binding.

  Carles Fernández-de-Alba, Nicholas S Berrow, Raquel Garcia-Castellanos, Jesús García, Miquel Pons
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  ABSTRACT: The nucleoid-associated protein H-NS is a global modulator of the expression of genes associated with adaptation to environmental changes. A variant of H-NS expressed in the R27 plasmid was previously shown to selectively modulate the expression of horizontally acquired genes, with minimal effects on core genes that are repressed by the chromosomal form of H-NS. Both H-NS proteins are formed by an oligomerization domain and a DNA-binding domain, which are connected by a linker that is highly flexible in the absence of DNA. We studied DNA binding by means of oligomer-forming chimeric proteins in which domains of the chromosomal and plasmidic variants are exchanged, as well as in monomeric truncated forms containing the DNA-binding domain and variable portions of the linker. Point mutations in the linker were also examined in full-length and truncated H-NS constructs. These experiments show that the linker region contributes to DNA binding affinity and that it is a main component of the distinct DNA binding properties of chromosomal and plasmidic H-NS. We propose that interactions between the linker and DNA limit the flexibility of the connection between H-NS oligomerization and DNA binding and provide an allosteric indirect readout mechanism to detect long-range distortions of DNA, thus enabling discrimination between core and horizontally acquired DNA.
  Journal of Molecular Biology 03/2013; · 4.00 Impact Factor
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  Article: Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism

  Yolanda Perez, Mariano Maffei, Ana Igea, Irene Amata, Margarida Gairí, Angel R Nebreda, Pau Bernadó, Miquel Pons
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  ABSTRACT: c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.
  Scientific Reports 02/2013; 3.
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  Available from: Miquel Pons

  Dataset: Thormann TOAIJ 2007

  Michael Thormann, David Vidal, Michael Almstetter, Miquel Pons
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  Available from: Miquel Pons

  Article: Oligomerization and DNA binding of Ler, a master regulator of pathogenicity of enterohemorrhagic and enteropathogenic Escherichia coli.

  Jesús García, Tiago N Cordeiro, María J Prieto, Miquel Pons
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  ABSTRACT: Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 3-5 units of Ler. DNA wraps around Ler particles that contain more than 15-16 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.
  Nucleic Acids Research 09/2012; · 8.03 Impact Factor