Fatty Acids Derived from Royal Jelly Are Modulators of Estrogen Receptor Functions

Royal jelly (RJ) excreted by honeybees and used as a nutritional and medicinal agent has estrogen-like effects, yet the compounds mediating these effects remain unidentified. The possible effects of three RJ fatty acids (FAs) (10-hydroxy-2-decenoic-10H2DA, 3,10-dihydroxydecanoic-3,10DDA, sebacic acid-SA) on estrogen signaling was investigated in various cellular systems. In MCF-7 cells, FAs, in absence of estradiol (E2), modulated the estrogen receptor (ER) recruitment to the pS2 promoter and pS2 mRNA levels via only ERβ but not ERα, while in presence of E2 FAs modulated both ERβ and ERα. Moreover, in presence of FAs, the E2-induced recruitment of the EAB1 co-activator peptide to ERα is masked and the E2-induced estrogen response element (ERE)-mediated transactivation is inhibited. In HeLa cells, in absence of E2, FAs inhibited the ERE-mediated transactivation by ERβ but not ERα, while in presence of E2, FAs inhibited ERE-activity by both ERβ and ERα. Molecular modeling revealed favorable binding of FAs to ERα at the co-activator-binding site, while binding assays showed that FAs did not bind to the ligand-binding pocket of ERα or ERβ. In KS483 osteoblasts, FAs, like E2, induced mineralization via an ER-dependent way. Our data propose a possible molecular mechanism for the estrogenic activities of RJ's components which, although structurally entirely different from E2, mediate estrogen signaling, at least in part, by modulating the recruitment of ERα, ERβ and co-activators to target genes

Multiple pH Regime Molecular Dynamics Simulation for pK Calculations

Ionisation equilibria in proteins are influenced by conformational flexibility, which can in principle be accounted for by molecular dynamics simulation. One problem in this method is the bias arising from the fixed protonation state during the simulation. Its effect is mostly exhibited when the ionisation behaviour of the titratable groups is extrapolated to pH regions where the predetermined protonation state of the protein may not be statistically relevant, leading to conformational sampling that is not representative of the true state. In this work we consider a simple approach which can essentially reduce this problem. Three molecular dynamics structure sets are generated, each with a different protonation state of the protein molecule expected to be relevant at three pH regions, and pK calculations from the three sets are combined to predict pK over the entire pH range of interest. This multiple pH molecular dynamics approach was tested on the GCN4 leucine zipper, a protein for which a full data set of experimental data is available. The pK values were predicted with a mean deviation from the experimental data of 0.29 pH units, and with a precision of 0.13 pH units, evaluated on the basis of equivalent sites in the dimeric GCN4 leucine zipper

How β-Lactam Antibiotics Enter Bacteria: A Dialogue with the Porins

BACKGROUND:Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. beta-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression. METHODOLOGY/PRINCIPAL FINDINGS: Here influx of beta-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single beta-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of beta-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility. CONCLUSIONS/SIGNIFICANCE: We propose the idea of a molecular "passport" that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections

MD simulation (allC, <b>Table 1</b>) of salt bridge connectivity of Glu11.

<p>The salt bridge partners are indicated in the panels.</p

Difference between p<i>K</i>_1/2 values calculated for chain A and chain B of GCN4 leucine zipper.

<p>Squares: calculations based on X-ray structure; Circles: calculations based on MD simulation. The upper and lower dashed lines correspond to |Δp<i>K</i>|<i>_av</i> = 1.2 (X-ray) and |Δp<i>K</i>|<i>_av</i> = 0.13 (MD), respectively.</p

Comparison of the p<i>K</i>_1/2 values calculated in this work with the experimental values.

a)<p>Calculation performed with the set of conformers when all titratable groups are charged (allC, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020116#pone-0020116-t001" target="_blank">Table 1</a>).</p>b)<p>Calculation performed with weighted contribution of all sets.</p>c)<p>Experimental data taken from Matousek et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020116#pone.0020116-Mitra1" target="_blank">[29]</a>.</p

Comparison of the calculated p<i>K</i>_1/2 values with the experimental data.

<p>Squares: calculations based on X-ray structure; Filled circles: calculations performed with weighted contribution of all sets; Open circles: calculations performed with the set of conformers when all titratable groups are charged (allC, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020116#pone-0020116-t001" target="_blank">Table 1</a>). The horizontal dashed lines at 0.29 and 0.87 mark the mean deviation of the calculated p<i>K</i> values on the basis of MD simulation and X-ray single structure, respectively from the experimental p<i>K</i> values. The continuous horizontal line delimits the precision of the calculations (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020116#pone-0020116-g001" target="_blank">Fig. 1</a>, Δp<i>K</i>|<i>_av</i>).</p

Schematic drawing of the environment of Glu11.

<p>The distances (Å) are averaged from the trajectory of the allC set. Letters A and B denote the corresponding chains of GCN4 leucine zipper. Values in parentheses are the standard deviations.</p

Electrostatic properties of the anion selective porin Omp32 from Delftia acidovorans and of the arginine cluster of bacterial porins

The functional properties of the anion-selective porin Omp32 from the bacterium Delftia acidovorans, formerly Comamonas acidovorans, are determined by the particularly narrow channel constriction and the electrostatic field inside and outside the pore. A cluster of arginines (Arg 38, Arg 75, and Arg 133) determines the electrostatic field close to the constriction zone. Stacked amino acids carrying charges are prone to drastic pKa shifts. However, optimized calculations of the titration behavior of charged groups, based on the finite-difference Poisson-Boltzmann technique, suggest that all the arginines are charged at physiological pH. Protonation of the clustered arginines is stabilized by one buried glutamate residue (Glu 58), which is strongly interacting with Arg 75 and Arg 38. This functional arrangement of three charged amino acid residues is of general significance because it is found in the constriction zones of all known 16-stranded porins from the α-, β-, and γ-proteobacteria

Understanding the −C–X1–X2–C– Motif in the Active Site of the Thioredoxin Superfamily: <i>E. coli</i> DsbA and Its Mutants as a Model System

<i>E. coli</i> DsbA is an intensively studied enzyme of the thioredoxin superfamily of thiol-disulfide oxidoreductases. DsbA catalyzes the disulfide bond formation and folding of proteins in the bacterial periplasm. DsbA and its mutants have highlighted the strong and puzzling influence of the −C–X1–X2–C– active site variants, found across the thioredoxin superfamily, on the ionization and redox properties of this site. However, the interpretation of these observations remains wanting, largely due to a dearth of structural information. Here, molecular dynamics simulations are used to provide extensive information on the structure and dynamics of reduced −C30–X31–X32–C33– motifs in wild type DsbA and 13 of its mutants. These simulations are combined with calculations of the p<i>K</i> of H32 and of the very low p<i>K</i> of the catalytic cysteine C30. In wild type DsbA, the titrations of C30 and H32 are shown to be coupled; the protonation states and dynamics of H32 are examined. The thiolate of C30 is stabilized by hydrogen bonds with the protein. Modulation of these hydrogen bonds by alteration of residue X32 has the greatest impact on the p<i>K</i> of C30, which rationalizes its higher p<i>K</i> in thioredoxin and tryparedoxin. Because of structural constrains, residue X31 has only an indirect and weak influence on the p<i>K</i> of C30. The dynamics of C30 is clearly related to its stabilizing interactions and p<i>K</i> value. Although relatively small differences between p<i>K</i>s were not reproduced in the calculations, the major trends are explained, adding new insights to our understanding of enzymes in this family