Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein

<div><p>Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.</p></div

Expression Analysis of Taste Signal Transduction Molecules in the Fungiform and Circumvallate Papillae of the Rhesus Macaque, <em>Macaca mulatta</em>

<div><p>The molecular mechanisms of the mammalian gustatory system have been examined in many studies using rodents as model organisms. In this study, we examined the mRNA expression of molecules involved in taste signal transduction in the fungiform papillae (FuP) and circumvallate papillae (CvP) of the rhesus macaque, <em>Macaca mulatta</em>, using <em>in situ</em> hybridization. <em>TAS1R1</em>, <em>TAS1R2, TAS2Rs, and PKD1L3</em> were exclusively expressed in different subsets of taste receptor cells (TRCs) in the FuP and CvP. This finding suggests that TRCs sensing different basic taste modalities are mutually segregated in macaque taste buds. Individual <em>TAS2Rs</em> exhibited a variety of expression patterns in terms of the apparent level of expression and the number of TRCs expressing these genes, as in the case of human <em>TAS2Rs</em>. <em>GNAT3</em>, but not <em>GNA14</em>, was expressed in TRCs of FuP, whereas <em>GNA14</em> was expressed in a small population of TRCs of CvP, which were distinct from <em>GNAT3</em>- or <em>TAS1R2</em>-positive TRCs. These results demonstrate similarities and differences between primates and rodents in the expression profiles of genes involved in taste signal transduction.</p> </div

Dimeric structure of His11Tyr mutant under neutral condition.

<p>A. Hydrogen bond interactions shown as dotted lines at the interface. B. Time dependent distances between Tyr11_A and Asp91_B, His14_A and Tyr21_B and Ser15_A and Gln35_B of the mutant under neutral conditions in red, purple and green traces, respectively. C. RMSD values for the main chain atoms of the His11Tyr mutant the 10 ns MD trajectory from the initial structure under the neutral condition (left) and the acidic condition (right). Line color is as defined for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126921#pone.0126921.g002" target="_blank">Fig 2B</a>.</p

The co-expression relationships among taste receptors and G protein α subunits.

<p>(A) In the CvP, <i>GNA14</i> was expressed in a much smaller population of TRCs than <i>GNAT3</i> and in a mutually exclusive manner. The <i>GNA14</i>–positive TRCs were distinct from those expressing <i>TAS1R2</i> and <i>TAS2R13</i>, but they were subsets of the <i>TAS1R3</i>-positive TRCs and partially overlapped with the <i>TAS1R1</i>-positive TRCs. n ≥1 (numbers of sections ≥2). (B) In the CvP, <i>TAS1R2</i> and <i>TAS2R13</i> were expressed in subsets of the <i>GNAT3</i>–positive TRCs, which partially overlapped with the <i>TAS1R1</i>- and <i>TAS1R3</i>-positive TRCs. n ≥2 (numbers of sections ≥4). (C) In the FuP, <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, and <i>TAS2R13</i> were expressed in subsets of <i>GNAT3</i>-positive TRCs. n ≥1 (numbers of sections ≥10). (D) Venn diagram illustrating the co-expression relationships among taste receptors and signal transduction molecules in the macaque and the mouse. Scale bars: 50 µm.</p

The mRNA expression of genes encoding taste receptors and signal transduction molecules in the fungiform and circumvallate papillae of the rhesus macaque.

<p>(A) <i>In situ</i> hybridization revealed that three <i>TAS1Rs</i>, <i>TAS2R13</i>, <i>PKD1L3</i>, <i>GNAT3</i>, <i>GNA14</i>, and <i>PLCB2</i> were robustly expressed in subsets of the TRCs in the CvP. These genes, except for <i>GNA14</i>, were also expressed in subsets of the TRCs in the FuP. n≥2 (numbers of sections ≥4) for <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, <i>PKD1L3</i>, <i>GNAT3</i>, and <i>TAS2R13</i> in CvP, n = 1 (numbers of sections ≥2) for <i>GNA14</i> and <i>PLCB2</i> in CvP, n≥2 (numbers of sections ≥20) for <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, <i>PKD1L3</i>, and <i>GNA14</i> in FuP, n = 1 (numbers of sections ≥10) for <i>GNAT3</i>, <i>PLCB2</i>, and <i>TAS2R13</i> in FuP. (B) The <i>TAS2Rs</i> located on chromosome 11 (<i>TAS2R9</i> and <i>TAS2R12-25</i>) appeared to be robustly expressed in subsets of TRCs, whereas only weak signals were observed for the <i>TAS2Rs</i> located on chromosomes 3 (<i>TAS2R1-8</i> and <i>TAS2R10-11</i>) and 6 (<i>TAS2R26</i>). <i>Tas2Rs</i> are arranged according to the locations on the chromosomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045426#pone.0045426.s001" target="_blank">Figure S1</a>). n = 2 (numbers of sections ≥4) for <i>TAS2R1-8</i>, <i>10</i>-<i>11</i>, <i>21</i>, <i>23</i>, and <i>26</i>, n = 1 (numbers of sections ≥2) for <i>TAS2R9</i>, <i>12, 14</i>-<i>20</i>, <i>22</i>, and <i>24</i>-<i>25</i>. Scale bars: 50 µm.</p

Dimeric interface of His11Phe mutant.

<p>Dotted lines show hydrogen bonds at the interface.</p

The co-expression relationships among taste receptors.

<p>(A) In the CvP, the <i>TAS1R3</i>-positive TRCs were negative for <i>TAS2R13</i>. The <i>PLCB2</i>-positive TRCs, which include <i>TAS1R1</i>-, <i>TAS1R2</i>-, <i>TAS1R3</i>-, and <i>TAS2R13</i>-positive TRCs, were negative for <i>PKD1L3</i>. n = 1 (numbers of sections ≥2). (B) In the FuP, the <i>TAS1R3</i>-positive TRCs were negative for <i>TAS2R13</i>. The <i>PLCB2</i>-positive TRCs, which include <i>TAS1R1</i>-, <i>TAS1R2</i>-, <i>TAS1R3</i>-, and <i>TAS2R13</i>-positive TRCs, were negative for <i>PKD1L3</i>. n = 1 or 2 (numbers of sections ≥10). Scale bars: 50 µm.</p

Dissociation of neoculin oligomer in MD calculations at acidic pH.

<p>A. An early stage (2 ns). B. A late stage (10 ns). Line color is as defined for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126921#pone.0126921.g011" target="_blank">Fig 11</a>. C. Distance between the centroids of each monomer in the 10 ns MD trajectory. Red, blue, purple and orange lines to represent the centroid distances between pairs of monomers represented in green and grey (GG), green and black (GB), green and yellow (GY), and yellow and black (YB), respectively.</p

Tetrameric structure of neoculin monomers in the crystal structure.

<p>A. The surfaces of Pro103A, Leu106A and Asn44B are colored orange. Another monomer (yellow ribbon) has contact with neoculin near these three residues. The other two neoculin molecules are shown as a grey surface and a black ribbon. B. Tetrameric structure rotated 90° clockwise from the structure depicted in A (left). Rotation of the structure on the left 180° about a vertical axis to show the structures on the other side of the dimer (right).</p

Location of residues showing mobile main chains on the neoculin monomer.

<p>A. The mobile residues are colored in red based on the difference in RMSD value for the main chain atoms of each residue in the MD trajectory. Residues in NAS and NBS are indicated by A and B with the residue number, respectively. B. The difference in RMSD value for the main chain atoms of each residue in the MD trajectory. Bars for mobile residues with RMSD values of ≥0.1 are colored in red and immobile residues with RMSD values of <0.1 are colored in grey. The regions of secondary structures are indicated in blue below the graph, and the yellow-colored regions are loop structures.</p