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The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor.

Winnig M, Bufe B, Kratochwil NA, Slack JP, Meyerhof W - BMC Struct. Biol. (2007)

Bottom Line: Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate.Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Institute of Human Nutrition Potsdam-Rehbruecke, Department of Molecular Genetics, Arthur-Scheunert Allee 114-116, 14558 Nuthetal, Germany. marcel.winnig.mw@axxam.com

ABSTRACT

Background: Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone.

Results: By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.

Conclusion: From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate. Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

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Alignment of the heptahelical domain of hCaSR, hmGlu2, rmGlu1, rmGlu5, and hTAS1R3. Transmembrane segments (TM) are shown in bold. Intra- and extracellular loops are marked by ICL and ECL, respectively. Amino acids that influence allosteric modulator activity in hCaSR, hmGlu2, rmGlu1 and 5 are marked in grey. Positions that alter the response of the sweet receptor to lactisole, cyclamate, or NHDC are shown in red. Asterisks indicate residues involved in receptor activation by NHDC (orange) or cyclamate (blue). Green asteriks denote residues mediating sensitivity of the receptor to lactisole.
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Figure 5: Alignment of the heptahelical domain of hCaSR, hmGlu2, rmGlu1, rmGlu5, and hTAS1R3. Transmembrane segments (TM) are shown in bold. Intra- and extracellular loops are marked by ICL and ECL, respectively. Amino acids that influence allosteric modulator activity in hCaSR, hmGlu2, rmGlu1 and 5 are marked in grey. Positions that alter the response of the sweet receptor to lactisole, cyclamate, or NHDC are shown in red. Asterisks indicate residues involved in receptor activation by NHDC (orange) or cyclamate (blue). Green asteriks denote residues mediating sensitivity of the receptor to lactisole.

Mentions: The sweet inhibitor lactisole, which interacts with specific residues in the heptahelical domain of TAS1R3 [13,15,16], inhibited the activation of the sweet receptor by NHDC and cyclamate competitively (Fig. 2). These results suggest that the binding sites of lactisole, cyclamate, and NHDC overlap. Interestingly, the observed competitive effects between NHDC and lactisole (Fig. 2) may also explain why lactisole in contrast to many other sweeteners did not inhibit the sweet taste of NHDC in humans [31]. A previous report of Jiang et al. proposed that cyclamate and lactisole interact with a common set of amino acid residues[14,15]. Our findings confirm this observation by showing that seven mutations (Q6373.29, H6413.33, W7756.48, F7786.51, L7826.55, R790Qex3, and C8017.39) influence both, the lactisole mediated inhibition and the cyclamate induced activation of the sweet receptor (Fig. 5, Table 1 + 2). While cyclamate and lactisole only use parts of the TAS1R3 pharmacophore, our model predicts that NHDC uses most of it due to its larger size (Fig. 3). In line with these predictions, eight of the seventeen amino acids that alter receptor activation by NHDC (Q6373.29, S6403.32, H6413.33, Y6994.60, W7756.48, F7786.51, L7826.55, and C8017.39), also influence lactisole-mediated inhibition of the receptor. Similarly, nine of the seventeen residues (Q6373.29, H6413.33, H721ex2, S7265.39, F7305.43, W7756.48, F7786.51, L7826.55, and C8017.39) mediate activation by cyclamate, while six (Q6373.29, H6413.33, W7756.48, F7786.51, L7826.55, and C8017.39) influence receptor inhibition by lactisole as well as receptor activation by cyclamate (Fig. 5, Table 1 + 2).


The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor.

Winnig M, Bufe B, Kratochwil NA, Slack JP, Meyerhof W - BMC Struct. Biol. (2007)

Alignment of the heptahelical domain of hCaSR, hmGlu2, rmGlu1, rmGlu5, and hTAS1R3. Transmembrane segments (TM) are shown in bold. Intra- and extracellular loops are marked by ICL and ECL, respectively. Amino acids that influence allosteric modulator activity in hCaSR, hmGlu2, rmGlu1 and 5 are marked in grey. Positions that alter the response of the sweet receptor to lactisole, cyclamate, or NHDC are shown in red. Asterisks indicate residues involved in receptor activation by NHDC (orange) or cyclamate (blue). Green asteriks denote residues mediating sensitivity of the receptor to lactisole.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2099433&req=5

Figure 5: Alignment of the heptahelical domain of hCaSR, hmGlu2, rmGlu1, rmGlu5, and hTAS1R3. Transmembrane segments (TM) are shown in bold. Intra- and extracellular loops are marked by ICL and ECL, respectively. Amino acids that influence allosteric modulator activity in hCaSR, hmGlu2, rmGlu1 and 5 are marked in grey. Positions that alter the response of the sweet receptor to lactisole, cyclamate, or NHDC are shown in red. Asterisks indicate residues involved in receptor activation by NHDC (orange) or cyclamate (blue). Green asteriks denote residues mediating sensitivity of the receptor to lactisole.
Mentions: The sweet inhibitor lactisole, which interacts with specific residues in the heptahelical domain of TAS1R3 [13,15,16], inhibited the activation of the sweet receptor by NHDC and cyclamate competitively (Fig. 2). These results suggest that the binding sites of lactisole, cyclamate, and NHDC overlap. Interestingly, the observed competitive effects between NHDC and lactisole (Fig. 2) may also explain why lactisole in contrast to many other sweeteners did not inhibit the sweet taste of NHDC in humans [31]. A previous report of Jiang et al. proposed that cyclamate and lactisole interact with a common set of amino acid residues[14,15]. Our findings confirm this observation by showing that seven mutations (Q6373.29, H6413.33, W7756.48, F7786.51, L7826.55, R790Qex3, and C8017.39) influence both, the lactisole mediated inhibition and the cyclamate induced activation of the sweet receptor (Fig. 5, Table 1 + 2). While cyclamate and lactisole only use parts of the TAS1R3 pharmacophore, our model predicts that NHDC uses most of it due to its larger size (Fig. 3). In line with these predictions, eight of the seventeen amino acids that alter receptor activation by NHDC (Q6373.29, S6403.32, H6413.33, Y6994.60, W7756.48, F7786.51, L7826.55, and C8017.39), also influence lactisole-mediated inhibition of the receptor. Similarly, nine of the seventeen residues (Q6373.29, H6413.33, H721ex2, S7265.39, F7305.43, W7756.48, F7786.51, L7826.55, and C8017.39) mediate activation by cyclamate, while six (Q6373.29, H6413.33, W7756.48, F7786.51, L7826.55, and C8017.39) influence receptor inhibition by lactisole as well as receptor activation by cyclamate (Fig. 5, Table 1 + 2).

Bottom Line: Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate.Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Institute of Human Nutrition Potsdam-Rehbruecke, Department of Molecular Genetics, Arthur-Scheunert Allee 114-116, 14558 Nuthetal, Germany. marcel.winnig.mw@axxam.com

ABSTRACT

Background: Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone.

Results: By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.

Conclusion: From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate. Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

Show MeSH
Related in: MedlinePlus