Characterization of Putative Virulence Factors of Pseudomonas aeruginosa Strain RBS Isolated from a Saltern, Tunisia: Effect of Metal Ion Cofactors on the Structure and the Activity of LasB

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium able to survive in diverse environments such as soil, plants, freshwater, and seawater. P. aeruginosa can be an opportunistic pathogen to humans when their immune system is deficient. Its pathogenicity may be linked to the production of virulence factors. We isolated P. aeruginosa strain RBS from the saltern of Sfax in Tunisia. In this study, we characterized the halotolerance, antibiotic susceptibility, and some virulence factors of strain RBS. High NaCl concentrations inhibited growth and motility. However, biofilm formation was enhanced to protect bacteria against salt stress. Among the 18 antibiotics tested, quinolones and tetracycline showed a significant inhibitory effect on growth, motility, and biofilm formation of strain RBS. β-Lactams, however, did not have any inhibitory effect on neither bacterial growth nor motility. In some cases, resistance was due, in part, to biofilm formation. We also showed that RBS produces two proteases, LasB and AprA, which have been shown to be implicated in host infection. LasB was further characterized to study the role of metal ions in enzyme stability. It possesses two distinct metal ion-binding sites coordinating a calcium and a zinc ion. The effect of metal ion chelation was evaluated as well as substitutions of residues involved in metal ion binding. Impairing metal ion binding of LasB led to a loss of activity and a sharp decrease of stability. Our findings suggest that the binding of both metal ions is interdependent as the two metal ions’ binding sites are linked via a hydrogen bond network.

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The influence of metal-ion binding on the structure and surface composition of Sonic Hedgehog: a combined classical and hybrid QM/MM MD study

Physical Chemistry Chemical Physics ◽

10.1039/c6cp03960j ◽

2016 ◽

Vol 18 (32) ◽

pp. 22254-22265 ◽

Cited By ~ 7

Author(s):

Manuel Hitzenberger ◽

Thomas S. Hofer

Keyword(s):

Metal Ions ◽

Sonic Hedgehog ◽

Binding Sites ◽

Metal Ion ◽

Surface Composition ◽

Quantum Mechanical ◽

Ion Binding ◽

Metal Ion Binding ◽

Structural Impact

The interaction of metal ions with Shh binding-sites and their structural impact are assessed via classical and quantum mechanical simulations.

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Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

Biochemical Journal ◽

10.1042/bj20061168 ◽

2006 ◽

Vol 400 (3) ◽

pp. 385-392 ◽

Cited By ~ 4

Author(s):

Erdeni Bai ◽

Federico I. Rosell ◽

Bao Lige ◽

Marcia R. Mauk ◽

Barbara Lelj-Garolla ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Metal Ions ◽

Binding Site ◽

Metal Ion ◽

Association Constants ◽

Ion Binding ◽

Ferric Uptake Regulator ◽

Metal Ion Binding ◽

Dimerization Domain ◽

High Affinity

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.

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Computational approaches for de novo design and redesign of metal-binding sites on proteins

Bioscience Reports ◽

10.1042/bsr20160179 ◽

2017 ◽

Vol 37 (2) ◽

Cited By ~ 12

Author(s):

Gunseli Bayram Akcapinar ◽

Osman Ugur Sezerman

Keyword(s):

Metal Ions ◽

Metal Binding ◽

Binding Sites ◽

Metal Ion ◽

De Novo ◽

De Novo Design ◽

Ion Binding ◽

Chemical Transformations ◽

Metal Ion Binding ◽

Metal Binding Sites

Metal ions play pivotal roles in protein structure, function and stability. The functional and structural diversity of proteins in nature expanded with the incorporation of metal ions or clusters in proteins. Approximately one-third of these proteins in the databases contain metal ions. Many biological and chemical processes in nature involve metal ion-binding proteins, aka metalloproteins. Many cellular reactions that underpin life require metalloproteins. Most of the remarkable, complex chemical transformations are catalysed by metalloenzymes. Realization of the importance of metal-binding sites in a variety of cellular events led to the advancement of various computational methods for their prediction and characterization. Furthermore, as structural and functional knowledgebase about metalloproteins is expanding with advances in computational and experimental fields, the focus of the research is now shifting towards de novo design and redesign of metalloproteins to extend nature’s own diversity beyond its limits. In this review, we will focus on the computational toolbox for prediction of metal ion-binding sites, de novo metalloprotein design and redesign. We will also give examples of tailor-made artificial metalloproteins designed with the computational toolbox.

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Quadrupolar central transition (QCT) and 13C NMR competition studies of metal ion binding to ovotransferrin

Canadian Journal of Chemistry ◽

10.1139/v11-019 ◽

2011 ◽

Vol 89 (7) ◽

pp. 779-788 ◽

Cited By ~ 2

Author(s):

Jillian A. Saponja ◽

Hans J. Vogel

Keyword(s):

Metal Ions ◽

Binding Sites ◽

Metal Ion ◽

Antimicrobial Agents ◽

Chemical Shifts ◽

Ion Binding ◽

Metal Ion Binding ◽

Ion Competition ◽

Central Transition ◽

C Nmr

The transferrins are a family of relatively large bilobal proteins that play a major role in the transport of Fe3+, as well as several other physiological and nonphysiological metal ions. Transferrins can also act as antimicrobial agents, by tightly sequestering iron and making it unavailable for bacterial growth. Using a combination of quadrupolar central transition (QCT) metal ion NMR (27Al, 45Sc, 51V, and 71Ga) and 13C NMR, the binding and displacement of a variety of metal ions to ovotransferrin was studied through direct metal ion competition experiments. The metal ions investigated (Al3+, Co3+, Fe3+, Ga3+, In3+, Sc3+, Y3+, and VO2+) were of differing ionic radius and charge, thus allowing for an assessment of how these factors contribute to metal ion affinity. The competition for the N- and C-terminal metal ion binding sites on ovotransferrin was directly followed by metal ion QCT NMR. Moreover, 13C NMR was used to study the two protein-bound synergistic anions (13C-labeled carbonate), whose chemical shifts are distinct and dependent on the bound metal ion that is present in the binding sites. The observed order of decreasing affinity for the metal ions studied was Fe3+ ≈ In3+ ≥ Sc3+ ≥ Ga3+ > Al3+ > VO2+ > Y3+ ≥ Co3+. These results illustrate how a combination of multinuclear solution NMR methods can provide unique insights into the ligand binding properties of larger metalloproteins.

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DNA Cleavage byEcoRV Endonuclease: Two Metal Ions in Three Metal Ion Binding Sites†

Biochemistry ◽

10.1021/bi0499056 ◽

2004 ◽

Vol 43 (22) ◽

pp. 6841-6857 ◽

Cited By ~ 60

Author(s):

Nancy C. Horton ◽

John J. Perona

Keyword(s):

Metal Ions ◽

Binding Sites ◽

Dna Cleavage ◽

Metal Ion ◽

Ion Binding ◽

Metal Ion Binding ◽

Ion Binding Sites ◽

Metal Ion Binding Sites

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Probing Ribosome Structure by Europium-Induced RNA Cleavage

Biological Chemistry ◽

10.1515/bc.1999.032 ◽

1999 ◽

Vol 380 (2) ◽

pp. 243-251 ◽

Cited By ~ 18

Author(s):

S. Dorner ◽

A. Barta

Keyword(s):

Metal Ions ◽

Binding Sites ◽

Metal Ion ◽

Divalent Metal ◽

23S Rrna ◽

Ion Binding ◽

Cleavage Sites ◽

Metal Ion Binding ◽

Divalent Metal Ions ◽

Binding Pockets

AbstractDivalent metal ions are absolutely required for the structure and catalytic activities of ribosomes. They are partly coordinated to highly structured RNA, which therefore possesses high-affinity metal ion binding pockets. As metalion induced RNA cleavages are useful for characterising metal ion binding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA ofE. coli. The cleavage sites were identified by primer extension and compared to those previously identified for calcium, lead, magnesium, and manganese ions. Several Eu3+cleavage sites, mostly those at which a general metal ion binding site had been already identified, were identical to previously described divalent metal ions. Overall, the Eu3+cleavages are most similar to the Ca2+cleavage pattern, probably due to a similar ion radius. Interestingly, several cleavage sites which were specific for Eu3+were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, but not tetracycline and streptomycin, significantly reduced Eu3+cleavage efficiencies in the peptidyl transferase center. The identification of specific Eu3+binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of metal ion binding pockets at the ribosome's active center.

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Selective metal ion binding at the calcium-binding sites of the sea urchin extraembryonic coat protein hyalin

Biochemistry and Cell Biology ◽

10.1139/o89-119 ◽

1989 ◽

Vol 67 (11-12) ◽

pp. 808-812 ◽

Cited By ~ 4

Author(s):

John J. Robinson

Keyword(s):

Coat Protein ◽

Metal Ions ◽

Sea Urchin ◽

Binding Sites ◽

Metal Ion ◽

Divalent Cations ◽

Biologically Active ◽

Ion Binding ◽

Metal Ion Binding ◽

Competition Assays

The interaction of metal ions with the sea urchin extraembryonic coat protein hyalin was investigated. Hyalin, immobilized on nitrocellulose membrane, bound Ca2+ and this interaction was disrupted by ruthenium red and selective metal ions. The divalent cations Cd2+ and Mn2+, when present at a concentration of 30 μM, displaced hyalin-bound Ca2+. In competition assays, 1 mM Cd2+ or 3 mM Mn2+ were effective competitors with Ca2+ for binding to hyalin. Cobalt, at a concentration of 30 μM, was unable to displace protein-bound Ca2+, but was effective in competition assays at a concentration of at least 10 mM. Magnesium and the monovalent cation Cs+ were unable to disrupt Ca2+–hyalin interaction. Interestingly, Cd2+, Mn2+, and Co2+ mimicked the biological effects of Ca2+ on the hyalin self-association reaction. These results clearly demonstrate that the Ca2+-binding sites on hyalin can selectively accommodate other divalent cations in a biologically active configuration.Key words: calcium, metal ion, binding, hyalin.

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Circularly polarized luminiscence as a probe of metal ions binding sites in calmodulin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19913028 ◽

1991 ◽

Vol 56 (12) ◽

pp. 3028-3031 ◽

Cited By ~ 7

Author(s):

Nursen Coruh ◽

James P. Riehl

Keyword(s):

Metal Ions ◽

Binding Sites ◽

Metal Ion ◽

Ion Concentration ◽

Ion Binding ◽

Metal Ion Binding ◽

Circularly Polarized ◽

Ion Binding Sites ◽

Metal Ion Binding Sites ◽

Dissymmetry Ratio

Circularly polarized luminiscence (CPL) from dilute solutions of Tb(III) bound to the Ca-binding protein calmodulin is reported. The dissymmetry ratio, gem, at 543.5 nm can be monitored as a function of equivalents of metal ion concentration. Competitive and consecutive addition of Ca(II) versus Tb(III) yield results which are consistent with previous results that suggest that Tb(III) and Ca(II) have a preferred affinity for different metal-ion binding sites in this protein.

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