The ArcB Leucine Zipper Domain Is Required for Proper ArcB Signaling

The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we report that the ArcB protein segment covering residues 70–121, fulfills the molecular characteristics of a leucine zipper containing coiled coil structure. Also, mutational analyses of this segment reveal three different phenotypical effects to be distributed along the coiled coil structure of ArcB, demonstrating that this motif is essential for proper ArcB signaling

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity

Effect of mutations in the leucine zipper of ArcB on its phosphatase activity and their dominant phenotypes.

<p><b>A</b>) Strain ECL5003 [<i>arcB</i>^wt, λΦ(<i>cydA’-lacZ</i>)] carrying low copy plasmids with the <i>arcB</i> mutant variants was grown aerobically (solid bars) or anaerobically (empty bars) to mid-exponential growth phase (OD₆₀₀ ∼ 0.5), and the ß-Galactosidase activity was assayed and expressed in Miller units. The data are averages from three independent experiments and the standard deviations are indicated. <b>B</b>) Effect of Leu replacements on the phosphatase activity of ArcB. Strain ECL5004, transformed with low-copy plasmids carrying different <i>arcB</i> forms, was grown aerobically to mid-exponential growth phase (OD₆₀₀ ∼ 0.5), on minimal medium with pyruvate as sole carbon source, and ß-Galactosidase activity was assayed and expressed in Miller units. The data are averages from three independent experiments and the standard deviations are indicated.</p

Effect of DTT and ubiquinone-0 on the rate of ArcB net-phosphorylation.

<p>Membrane vesicles (1 µg) containing high amounts of wild type ArcB^1–778 (circles) or the mutant ArcB variants (ArcB^L80V (squares) and ArcB^L87V (diamonds)) were incubated at room temperature with 40 µM [γ-³²P]ATP in the presence of 5 mM DTT (open symbols) or 250 µM Q0 (closed symbols) in a 20 µl reaction mixture. At the indicated time intervals a 5 µl sample was withdrawn for SDS-PAGE analysis. Left panel: autoradiograms of the gels. Right panel: net increase of ArcB-P with time, as quantitated with a PhosphorImager.</p

Effect of mutations in the leucine zipper of ArcB on the expressions of λΦ(<i>cydA’-lacZ</i>) and λΦ(<i>lldP’- lacZ</i>) operon fusions.

<p><b>A</b>) Strains ECL5012 [λΦ(<i>lldP’- lacZ</i>)] and ECL5004 [λΦ(<i>cydA’-lacZ</i>)] carrying low copy plasmids that harbor the <i>arcB</i> mutant variants were grown aerobically (solid bars) or anaerobically (empty bars) in Luria-Bertani broth containing 0.1 M MOPS (morpholinepropanesulfonic acid; pH 7.4) and 20 mM D-xylose. In the case of the λΦ(<i>lldP’- lacZ</i>)-bearing strains the growth medium was supplemented with 20 mM L-lactate as an inducer. At mid-exponential growth phase (OD₆₀₀ ∼ 0.5) the cells were harvested and the ß-Galactosidase activity was assayed and expressed in Miller units. The data are averages from three independent experiments and the standard deviations are indicated. (<b>B</b>) Equal number of bacteria of the above aerobic cultures were analyzed by Western blot analysis, using ArcB polyclonal antibodies as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038187#pone.0038187-Kwon3" target="_blank">[34]</a>.</p

<i>Escherichia coli</i> strains and plasmids used in this study are listed.

<p><i>Escherichia coli</i> strains and plasmids used in this study are listed.</p

Electrophysiological properties of WT, PG6, and PA6 channels and their regulation by β2a and β3.

<p>The values of V₅₀ and k were calculated for each cell, then averaged. R values determined from test pulses to +20 mV. Data shown are mean±SEM from the number of cells shown in parentheses. Statistical significance of the β2a and β3 effects relative to either α₁ alone (+α₂δ1) were determined using ANOVA.</p>†<p>Currents from PG6 were completely inactivated by 350 ms, so the residual current at 25 ms (divided by peak) is reported (R₂₅).</p>§<p>Current density was estimated from the peak of the <i>I–V</i> curve, and statistical significance was determined using Student's <i>t</i>-test.</p><p>*P<0.05, **P<0.01.</p