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According to the grounding work of Jacob and Monod, transcription factors (TFs) regulate gene expression by modulating their DNA binding affinity in response to a particular signal, or in the presence of a specific cofactor, much akin inducible or repressible on-off switches. Whilst the vast majority of TFs recognize distinct elements through the readout of conserved nucleotide motifs in the major groove of DNA, a few are able to bind specifically in the minor groove. Here we report the finding of a TF able to discriminatively exploit the binding to either the major or the minor DNA groove in order to mediate opposite read-out of the same regulatory signal. This mechanism accounts for the ability of the ferric uptake regulator Fur, a widespread prokaryotic transcriptional regulator involved in metal ion homeostasis and virulence in many bacteria, to repress the expression of both iron-inducible and iron-repressible genes in the human pathogen Helicobacter pylori. In particular, we demonstrate that Fur exploits the two grooves of DNA to function like a molecular commutator switch. In the apo-form, Fur binds with higher affinity to an iron-induced promoter as dimer, through the readout of thymine dimers in the major groove, contributing to its transcriptional repression under iron-deplete conditions. Conversely, on iron-repressed promoters, the metal ion acts as co-repressor, inducing protein conformational changes that prompt multimerization of the regulator and high affinity binding to the minor groove in occurrence of AT-rich inverted repeats. The latter mechanism appears to involve an Arg residue in helix H1 of the HTH-DNA-binding domain. In addition, molecular modelling and in silico docking indicate that binding to the minor groove involves conformation of the HTH-DNA binding domains of four Fur monomers into a tetrameric DNA clamp. These results provide evidence for a novel regulatory mechanism that may be exploited also in other bacteria.

An integrated approach, combining atomistic molecular dynamics simulations, coarsegrained models, and solution NMR, was used to characterize the internal dynamics of HpNikR, a Ni-dependent transcription factor. Specifically, these methods were used to ascertain how the presence of bound Ni2+ ions affects the stability of the known open, cis, and trans forms observed in the crystal structures of this protein as well as their interconversion capability. The consensus picture emerging from all the collected data hints at the interconversion of NikR among the three types of conformations, regardless of the content of bound Ni2+. On the basis of atomistic and coarse-grained simulations, we deduce that the interconversion capability is particularly effective between the cis and the open forms and appreciably less so between the trans conformer and the other two forms. The presence of the bound Ni2+ ions does, however, affect significantly the degree of the correlations on the two DNA-binding domains of NikR, which is significantly suppressed as compared to the apo form. Overall, the findings suggest that the binding of HpNikR to DNA occurs through a sophisticated multistep process involving both a conformational selection and an induced fit.

Nickel is both a toxic and an essential component of the active site of several enzymes. Urease, a Ni2+-enzyme of high relevance for human health and agriculture, is the final destination of the greater part of the intracellular Ni2+. The mechanisms that regulate Ni2+ trafficking leading to urease activation represent a paradigm to understand the general principles of cellular Ni2+ homeostasis. NikR is a metal-sensor that responds to different concentrations of intracellular Ni2+, controlling the transcription of the urease operon: the latest insights into metal-binding and DNA-binding of the Ni2+ sensor NikR will be illustrated. The mechanism of Ni2+ delivery into the apo-urease precursor will be described on the basis of the available information on the urease accessory proteins UreD, UreE, UreF, and UreG, which mediate the insertion of nickel into the enzyme active site.

The essentiality of Ni2+ for urease activity demands proper intracellular Ni2+ trafficking. The metallo-chaperone UreE delivers Ni2+ into the apo-enzyme in the last step of urease maturation, concomitantly with GTP hydrolysis catalyzed by UreG. This study is focused on UreE and UreG from Helicobacter pylori, a ureolytic bacterium responsible for gastric ulcers and cancer. HpUreG tightly binds 0.5 equivalents of Zn2+ per monomer, whereas it has 20-fold lower affinity for Ni2+. Zn2+ binding (but not Ni2+ binding) causes HpUreG dimerization. The role of conserved protein residues in metal binding was verified by site-directed mutagenesis. HpUreE binds one equivalent of Ni2+ or Zn2+ per dimer, with protein conformation depending on the metal ion. A stable UreE-UreG protein complex, specifically induced by Zn2+ and not by Ni2+, was identified and a viable structure calculated. A possible role for Zn2+ in the Ni2+-dependent urease assembly process mediated by UreE and UreG is envisaged.

NikR is a nickel-dependent transcriptional factor that regulates the expression of genes coding for proteins involved in the modulation of intracellular concentration of nickel ions (Ni2+) in the pathogen Helicobacter pylori (Hp), and is crucial for its survival in human stomach. To investigate the mechanism through which Ni2+ affects the protein structural and dynamics properties and consequently its affinity towards DNA, different states of the protein (open, closed-trans and closed-cis) were modelled. They differ for the relative orientation of DNA Binding Domains (DBDs) according to the ACT domain (metal binding domain). For each state, different models considering Ni2+ presence in high affinity binding sites were constructed. Each model was subjected to 40 ns long molecular dynamics simulations in explicit water as well as to Poisson-Boltzmann calculations. The ACT domain turned out to rearrange differently depending on the presence or the absence of Ni2+ ions. Contraction of ACT domain during the simulation of the protein fully loaded with Ni2+ is achieved through formation of a hydrogen bond network. Changes of the ACT domain affect rearrangement of DBDs and, consequently, proteins ability to bind DNA. Furthermore, the presence of Ni2+ ions causes an increase of positive potential in the DNA binding region, consistent with the experimental fact that that the presence of the ions increases its affinity for DNA.

PR-10 proteins are a family of pathogenesis related (PR) allergenic proteins playing multifunctional roles. The peach (Prunus persica) major allergen, Pru p 1.01, and its isoform Pru p 1.06D were found highly expressed in the fruit skin at the pit hardening stage, when fruits transiently lose their susceptibility to the fungal pathogen Monilinia spp. To investigate the possible role of the two Pru p 1 isoforms in plant defense, the recombinant proteins were expressed in E. coli and purified. Even though the proteins do not display direct antimicrobial activity, they both act as RNases, a function possibly related to defense. The RNase activity is different for the two proteins, and only that of Pru p 1.01 is affected in the presence of the cytokinin zeatin, suggesting a physiological correlation between Pru p 1.01 ligand binding and enzymatic activity. The binding of zeatin to Pru p 1.01 was evaluated using isothermal titration calorimetry, which provided information on the stoichiometry and on the thermodynamic parameters of the interaction.

The glycolytic enzyme enolase has been identified as putative plasminogen (Plg) binding protein in B. lactis BI07. Since enolase represents one of the best characterized Plg receptors in prokaryotes and eukaryotes, here we characterized this B. lactis housekeeping enzyme with respect to surface localization and Plg binding activity. The display of enolase on B. lactis BI07 cell surface was proved by immunoelectron microscopy. In order to individuated the putative Plg binding sites of B. lactis enolase, an high quality homology model was generated. The B. lactis enolase possesses structural homologue of the internal Plg binding site BS2 of pneumococcal enolase, as well as a C-terminal lysine, which constitute the Plg binding site BS1. In order to characterize the mechanism of Plg binding of B. lactis enolase, the wild type protein, as well as the protein mutated in the BS2, BS1, and BS2/BS1 homologues, were purified and their Plg binding activity evaluated. According to our preliminary data, the homologue of BS2 represents an essential Plg binding site of B. lactis BI07 enolase whereas the homologue of BS1 is only marginally involved.

In oxygenic photosynthesis, plastocyanin shuttles electrons between the membrane-bound complexes cytochrome b6f and photosystem I. The homologous complex between cytochrome f and plastocyanin, both from spinach, is the object of this study. The solution structure of the reduced spinach plastocyanin was determined using high field NMR spectroscopy, while the model structure of oxidized cytochrome f was obtained by homology modeling calculations and molecular dynamics. The model structure of the intermolecular complex was calculated using the program AUTODOCK, taking into account biological information obtained from mutagenesis experiments. The best electron transfer pathway from the heme group of cytochrome f to the copper ion of plastocyanin was calculated using the program HARLEM, obtaining a coupling decay value of 1.8 x 10-4. Possible mechanisms of interaction and electron transfer between plastocyanin and cytochrome f were discussed considering the possible formation of a super-complex that associates one cytochrome b6f, one photosystem I, and one plastocyanin.