The Intracellular Loop of the Na⁺/Ca²⁺ Exchanger Contains an “Awareness Ribbon”-Shaped Two-Helix Bundle Domain

The Na⁺/Ca²⁺ exchanger (NCX) is a ubiquitous single-chain membrane protein that plays a major role in regulating the intracellular Ca²⁺ homeostasis by the counter transport of Na⁺ and Ca²⁺ across the cell membrane. Other than its prokaryotic counterpart, which contains only the transmembrane domain and is self-sufficient as an active ion transporter, the eukaryotic NCX protein possesses in addition a large intracellular loop that senses intracellular calcium signals and controls the activation of ion transport across the membrane. This provides a necessary layer of regulation for the more complex function of eukaryotic cells. The Ca²⁺ sensor in the intracellular loop is known as the Ca²⁺-binding domain (CBD12). However, how the signaling of the allosteric intracellular Ca²⁺ binding propagates and results in transmembrane ion transportation still lacks a detailed explanation. Further structural and dynamics characterization of the intracellular loop flanking both sides of CBD12 is therefore imperative. Here, we report the identification and characterization of another structured domain that is N-terminal to CBD12 in the intracellular loop using solution nuclear magnetic resonance (NMR) spectroscopy. The atomistic structure of this domain reveals that two tandem long α-helices, connected by a short linker, form a stable crossover two-helix bundle (THB), resembling an “awareness ribbon”. Considering the highly conserved amino acid sequence of the THB domain, the detailed structural and dynamics properties of the THB domain will be common among NCXs from different species and will contribute toward the understanding of the regulatory mechanism of eukaryotic Na⁺/Ca²⁺ exchangers

pH Dependence of the Stress Regulator DksA

<div><p>DksA controls transcription of genes associated with diverse stress responses, such as amino acid and carbon starvation, oxidative stress, and iron starvation. DksA binds within the secondary channel of RNA polymerase, extending its long coiled-coil domain towards the active site. The cellular expression of DksA remains constant due to a negative feedback autoregulation, raising the question of whether DksA activity is directly modulated during stress. Here, we show that <i>Escherichia coli</i> DksA is essential for survival in acidic conditions and that, while its cellular levels do not change significantly, DksA activity and binding to RNA polymerase are increased at lower pH, with a concomitant decrease in its stability. NMR data reveal pH-dependent structural changes centered at the interface of the N and C-terminal regions of DksA. Consistently, we show that a partial deletion of the N-terminal region and substitutions of a histidine 39 residue at the domain interface abolish pH sensitivity in vitro. Together, these data suggest that DksA responds to changes in pH by shifting between alternate conformations, in which competing interactions between the N- and C-terminal regions modify the protein activity.</p></div

DksA is sensitive to changes in pH.

<p>(A) DksA activity increases at low pH. Increasing concentrations of DksA were added to holo RNAP (30 nM), ApC dinucleotide (0.2 mM), UTP (0.2 mM), GTP (4 μM) and [α-³²P]-GTP (10 μCi of 3000 Ci mmol⁻¹) followed by incubation for 15 minutes in Transcription buffer (20 mM Tris-HCl pH 7.9, 20 mM NaCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA). A linear DNA fragment containing the <i>rrnB</i> P1 promoter was added to initiate transcription and the formation of a 4 nucleotide RNA product was monitored on a denaturing 8% acrylamide gel. A dotted line marks the inhibition of 50% of transcription and is denoted as IC₅₀. The IC₅₀ values (calculated using a single-site binding equation from three independent repeats combined in a best-fit curve, in μM) were: pH 7.6 − 0.7 ± 0.28, pH 6.7 − 0.11 ± 0.016. (B) DksA affinity to core increases at lower pH. DksA binding to core RNAP was performed using the localized Fe²⁺ mediated cleavage assay at different pH. DksA concentrations were: 0, 25, 50, 100, 200 and 400 nM. FL—Full length protein, Cl—cleaved protein, Kd app—apparent Kd.</p

Substitution of His39 alters DksA sensitivity to pH.

<p>(A) The effect of pH on DksA variants. DksA activity and IC₅₀ calculations were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g003" target="_blank">Fig. 3A</a> with the <i>rrnB</i> P1 promoter. Experiments were performed at least three times at each pH. (B) Thermostability of DksA^H39A is low and relatively insensitive to pH. Thermostability was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g004" target="_blank">Fig. 4</a>.</p

DksA structure is sensitive to pH.

<p>Two-dimensional ¹H-¹⁵N HSQC spectra at pH 8 (black) and 6 (red) reveal large chemical shift changes at (A) Tyr23 and (B) many other residues.</p

Δ<i>dksA</i> mutants are sensitive to acidic conditions.

<p>(A) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown overnight in rich medium at pH 7.8. Cultures were diluted 1:50 into LB medium at pH 2.5. At selected time points aliquots were taken and the percentage of survival of bacteria was determined using viable count. (B) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown at pH 7.8, followed by 2.5 hour adaptation at pH 6.5–4.5, and then diluted into LB medium at pH 3.5. Survival was determined using viable counts; the result after 2 hour incubation at pH 3.5 is shown. (C) DksA concentration remains relatively constant at low pH. Samples taken at different time points after a change in pH were analyzed using Western blotting with anti-DksA antibodies. Extract from the Δ<i>dksA</i> strain and purified DksA were loaded as controls.</p

Constituents of an Extract of <i>Cryptocarya rubra</i> Housed in a Repository with Cytotoxic and Glucose Transport Inhibitory Effects

A new alkylated chalcone (<b>1</b>), a new 1,16-hexadecanediol diester (<b>2</b>), and eight known compounds were isolated from a dichloromethane-soluble repository extract of the leaves and twigs of <i>Cryptocarya rubra</i> collected in Hawaii. The structures of the new compounds were determined by interpretation of their spectroscopic data, and the absolute configurations of the two known cryptocaryanone-type flavonoid dimers, (+)-bicaryanone A (<b>3</b>) and (+)-chalcocaryanone C (<b>4</b>), were ascertained by analysis of their electronic circular dichroism and NOESY NMR spectra. All compounds isolated were evaluated against HT-29 human colon cancer cells, and, of these, (+)-cryptocaryone (<b>5</b>) was found to be potently cytotoxic toward this cancer cell line, with an IC₅₀ value of 0.32 μM. This compound also exhibited glucose transport inhibitory activity when tested in a glucose uptake assay

Antioxidant and Quinone Reductase-Inducing Constituents of Black Chokeberry (Aronia melanocarpa) Fruits

Using in vitro hydroxyl radical-scavenging and quinone reductase-inducing assays, bioactivity-guided fractionation of an ethyl acetate-soluble extract of the fruits of the botanical dietary supplement, black chokeberry (Aronia melanocarpa), led to the isolation of 27 compounds, including a new depside, ethyl 2-[(3,4-dihydroxybenzoyloxy)-4,6-dihydroxyphenyl] acetate (<b>1</b>), along with 26 known compounds (<b>2</b>–<b>27</b>). The structures of the isolated compounds were identified by analysis of their physical and spectroscopic data ([α]_D, NMR, IR, UV, and MS). Altogether, 17 compounds (<b>1</b>–<b>4</b>, <b>9</b>, <b>15</b>–<b>17</b>, and <b>19</b>–<b>27</b>) showed significant antioxidant activity in the hydroxyl radical-scavenging assay, with hyperin (<b>24</b>, ED₅₀ = 0.17 μM) being the most potent. The new compound (<b>1</b>, ED₅₀ = 0.44 μM) also exhibited potent antioxidant activity in this assay. Three constituents of black chokeberry fruits doubled quinone reductase activity at concentrations <20 μM, namely, protocatechuic acid [<b>9</b>, concentration required to double quinone reductase activity (CD) = 4.3 μM], neochlorogenic acid methyl ester (<b>22</b>, CD = 6.7 μM), and quercetin (<b>23</b>, CD = 3.1 μM)

Bioassay-Guided Isolation of Antioxidant and Cytoprotective Constituents from a Maqui Berry (<i>Aristotelia chilensis</i>) Dietary Supplement Ingredient As Markers for Qualitative and Quantitative Analysis

Bioassay-guided phytochemical investigation of a commercially available maqui berry (<i>Aristotelia chilensis</i>) extract used in botanical dietary supplement products led to the isolation of 16 compounds, including one phenolic molecule, <b>1</b>, discovered for the first time from a natural source, along with several known compounds, <b>2</b>–<b>16</b>, including three substances not reported previously in <i>A. chilensis</i>, <b>2</b>, <b>14</b>, and <b>15</b>. Each isolate was characterized by detailed analysis of NMR spectroscopic and HRESIMS data and tested for their in vitro hydroxyl radical scavenging and quinone-reductase inducing biological activities. A sensitive and accurate LC–DAD-MS method for the quantitative determination of the occurrence of six bioactive compounds, <b>6</b>, <b>7</b>, <b>10</b>–<b>12</b>, and <b>14</b>, was developed and validated using maqui berry isolates purified in the course of this study as authentic standards. The method presented can be utilized for dereplication efforts in future natural product research projects or to evaluate chemical markers for quality assurance and batch-to-batch standardization of this botanical dietary supplement component

Computer-Assisted Structure Elucidation of Black Chokeberry (<i>Aronia melanocarpa</i>) Fruit Juice Isolates with a New Fused Pentacyclic Flavonoid Skeleton

Melanodiol 4″-<i>O</i>-protocatechuate (<b>1</b>) and melanodiol (<b>2</b>) represent novel flavonoid derivatives isolated from a botanical dietary supplement ingredient, dried black chokeberry (<i>Aronia melanocarpa</i>) fruit juice. These noncrystalline compounds possess an unprecedented fused pentacyclic core with two contiguous hemiketals. Due to having significant hydrogen deficiency indices, their structures were determined using computer-assisted structure elucidation software. The in vitro hydroxyl radical-scavenging and quinone reductase-inducing activity of each compound are reported, and a plausible biogenetic scheme is proposed