Identification of key amino acid residues in the hTGR5-nomilin interaction and construction of its binding model - PubMed (original) (raw)

Identification of key amino acid residues in the hTGR5-nomilin interaction and construction of its binding model

Takashi Sasaki et al. PLoS One. 2017.

Abstract

TGR5, a member of the G protein-coupled receptor (GPCR) family, is activated by bile acids. Because TGR5 promotes energy expenditure and improves glucose homeostasis, it is recognized as a key target in treating metabolic diseases. We previously showed that nomilin, a citrus limonoid, activates TGR5 and confers anti-obesity and anti-hyperglycemic effects in mice. Information on the TGR5-nomilin interaction regarding molecular structure, however, has not been reported. In the present study, we found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5). Using mouse-human chimeric TGR5, we also found that three amino acid residues (Q77ECL1, R80ECL1, and Y893.29) are important in the hTGR5-nomilin interaction. Based on these results, an hTGR5-nomilin binding model was constructed using in silico docking simulation, demonstrating that four hydrophilic hydrogen-bonding interactions occur between nomilin and hTGR5. The binding mode of hTGR5-nomilin is vastly different from those of other TGR5 agonists previously reported, suggesting that TGR5 forms various binding patterns depending on the type of agonist. Our study promotes a better understanding of the structure of TGR5, and it may be useful in developing and screening new TGR5 agonists.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Differences between hTGR5 and mTGR5 with respect to nomilin response and amino acid sequences.

(A) Structural representation of nomilin. (B) HEK293 cells were transfected with the CRE-driven luciferase reporter plasmid and the hTGR5/mTGR5 expression plasmid. After transfection for 24 h, the cells were treated with TLCA (positive control) and nomilin (100 μM each) for another 5 h. Then, a luciferase reporter assay was performed, normalizing against β-galactosidase activity. The promoter activity of hTGR5/DMSO was set at 1 (n = 3). (C) Amino acid-sequence alignment and TM domain of the hTGR5 and mTGR5; unconserved amino acids are shown in inverted color. Significant differences were analyzed using one-way ANOVA (Tukey’s post hoc test); **p < 0.01. The values represent the mean ± SD.

Fig 2

Fig 2. Nomilin and TLCA specificity of chimeric TGR5.

(A–D) Left panels show diagrammatic representations of chimeric TGR5. hTGR5 is shown in beige with yellow TM domains, and mTGR5 is shown in blue with green TM domains. Right panels show CRE-Luc response profiles to nomilin and TLCA (100 μM each) for corresponding hTGR5, mTGR5, and chimeric TGR5. Significant differences between the nomilin responses were analyzed using one-way ANOVA (Tukey’s post hoc test); **p < 0.01 for mTGR5-Nomilin; #p < 0.05 and ##p < 0.01 for hTGR5-Nomilin. The values represent the mean ± SD.

Fig 3

Fig 3. Response profiles of multiple mouse-to-human point mutations in nomilin and TLCA.

Transient transfection assays using HEK293 cells with a CRE-luciferase reporter plasmid and expression vector for TGR5 with indicated point mutation of nine unconserved residues in ECL2 (A) and TM3 (B). After transfection for 24 h, the cells were treated with TLCA and nomilin (100 μM each) for another 5 h. Then, luciferase reporter activities were quantified (n = 3). Significant differences between the nomilin responses were analyzed using one-way ANOVA (Tukey’s post hoc test); **p < 0.01 for mTGR5-WT. The values represent the mean ± SD.

Fig 4

Fig 4. Differences in the affinity between hTGR5 and mTGR5 for nomilin as determined using three amino acid residues.

(A) Transient transfection assays using HEK293 cells with a CRE-luciferase reporter plasmid and an expression vector for TGR5 with the indicated mutation of three unconserved residues (n = 3). (B) Each WT and mutant TGR5 was transfected into HEK293 cells together with the CRE-luciferase plasmid, and dose-response curves to nomilin (left panel) and TLCA (right panel) were examined. The lower panel shows the EC50 values of each TGR5 for nomilin and TLCA (n = 3). Significant differences between the nomilin responses were analyzed using one-way ANOVA (Tukey’s post hoc test); **p < 0.01 and *p < 0.05 for hTGR5-WT; ##p < 0.01 and #p < 0.05 for mTGR5-WT. The values represent the mean ± SD.

Fig 5

Fig 5. Binding modes and docking interactions of nomilin with hTGR5.

(A and B) Hydrophilic hydrogen-bonding interaction between nomilin and three amino acid residues of hTGR5 are shown with red dashes.

Fig 6

Fig 6. Obacunone activates hTGR5 in a binding pattern analogous to nomilin.

(A) Structural representation of obacunone. (B) Comparison between the binding pattern of hTGR5 and those of obacunone (cyan) and nomilin (yellow). (C) Hydrophilic hydrogen-bonding interaction and CH-π interaction occurring between obacunone and three amino acid residues of hTGR5 are shown in red- and blue-dashed lines, respectively. (D) HEK293 cells were transfected with the CRE-driven luciferase reporter plasmid and the hTGR5 or mTGR5 expression plasmid. After transfection for 24 h, the cells were treated with TLCA, nomilin, and obacunone (100 μM each) for another 5 h. Then, the luciferase reporter activity was quantified. Significant differences were analyzed using one-way ANOVA (Tukey’s post hoc test); **p < 0.01. (E) Response profiles to DMSO, obacunone, and TLCA indicated for TGR5. Significant differences between the obacunone responses were analyzed using one-way ANOVA (Tukey’s post hoc test); **p < 0.01 for hTGR5-WT; ##p < 0.01 for mTGR5-WT. (F) Each WT and mutant TGR5 was transfected into HEK293 cells together with CRE-luciferase plasmid, and the dose-response curves to obacunone were examined. The right panel shows the EC50 of each TGR5 for the obacunone. The values represent the mean ± SD.

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Grants and funding

This study was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 15H05781 to R.S., No. 16K18699 to T.S.), the Cross-ministerial Strategic Innovation Promotion Program (No. 14533567 to R.S.), and the Japanese Agency for Medical Research and Development (AMED-CREST, No. 16gm0910008h0001 to R.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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