Thermostabilization of the Human Endothelin Type B Receptor

Okuta, Akiko; Tani, Kazuhide; Nishimura, Shoko; Fujiyoshi, Yoshinori; Doi, Tomoko

doi:10.1016/j.jmb.2016.03.024

Cited by 16 publications

(14 citation statements)

References 26 publications

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“…For crystallization, we used the previously established, thermostabilized ET B receptor (ET B -Y4) 22,23 . The IC 50 value of IRL2500 for ET B -Y4 was similar to that for the wild type receptor in the TGFα shedding assay 24 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of human endothelin ETB receptor in complex with peptide inverse agonist IRL2500

et al. 2019

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Endothelin receptors (ET A and ET B ) are G-protein-coupled receptors activated by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide-mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a potent ET B -selective antagonist, which has preventive effects against brain edema. Here, we report the crystal structure of the human ET B receptor in complex with IRL2500 at 2.7 Å-resolution. The structure revealed the different binding modes between IRL2500 and endothelin-1, and provides structural insights into its ET B -selectivity. Notably, the biphenyl group of IRL2500 penetrates into the transmembrane core proximal to D 2.50 , thus stabilizing the inactive conformation. Using the newly-established constitutively active mutant, we clearly demonstrate that IRL2500 functions as an inverse agonist for the ET B receptor. The current findings will expand the chemical space of ETR antagonists and facilitate the design of inverse agonists for other class A GPCRs.

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Section: Resultsmentioning

confidence: 99%

Crystal structure of human endothelin ETB receptor in complex with peptide inverse agonist IRL2500

et al. 2019

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“…To obtain mechanistic insights into the different actions of these agonists, we performed X-ray crystal structural analyses of the human ET B receptor in complex with ET-3 and IRL1620. For crystallization, we used the previously established, thermostabilized ET B receptor (ET B -Y5) 27 , 30 . IRL1620 also functions as a partial agonist for the thermostabilized receptor, as the E max values for ET B -Y5 were lower than those of ET-1 in both assays (84% and 85% in the TGFα shedding assay and the β-arrestin recruitment assay, respectively), while the EC 50 values of IRL1620 were increased for ET B -Y5 by about 9- and 6-fold in the TGFα shedding assay and β-arrestin recruitment assay, respectively, as compared to the wild type receptor (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation

et al. 2018

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Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.

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“…Here we introduced a new comprehensive CompoMug approach for the prediction of stabilizing mutations in GPCRs and demonstrated its first application. In the 5-HT 2C case, CompoMug achieved a very high success rate of 25% for mutations with substantial, more than 1.5°C, improvement in Tm values, which is 5 to 15 times higher than corresponding hit rates found in the experimental alanine mutagenesis approach for the adenosine or endothelin type B receptors ( Lebon et al, 2011 ; Okuta et al, 2016 ). The CompoMug predictions resulted in the discovery of 10 new stabilizing mutations and enabled the structure determination of the 5-HT 2C receptor in complexes with an agonist and an antagonist.…”

Section: Discussionmentioning

confidence: 82%

Computational design of thermostabilizing point mutations for G protein-coupled receptors

Popov

Peng

Shen

et al. 2018

eLife

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Engineering of GPCR constructs with improved thermostability is a key for successful structural and biochemical studies of this transmembrane protein family, targeted by 40% of all therapeutic drugs. Here we introduce a comprehensive computational approach to effective prediction of stabilizing mutations in GPCRs, named CompoMug, which employs sequence-based analysis, structural information, and a derived machine learning predictor. Tested experimentally on the serotonin 5-HT2C receptor target, CompoMug predictions resulted in 10 new stabilizing mutations, with an apparent thermostability gain ~8.8°C for the best single mutation and ~13°C for a triple mutant. Binding of antagonists confers further stabilization for the triple mutant receptor, with total gains of ~21°C as compared to wild type apo 5-HT2C. The predicted mutations enabled crystallization and structure determination for the 5-HT2C receptor complexes in inactive and active-like states. While CompoMug already shows high 25% hit rate and utility in GPCR structural studies, further improvements are expected with accumulation of structural and mutation data.

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Thermostabilization of the Human Endothelin Type B Receptor

Cited by 16 publications

References 26 publications

Crystal structure of human endothelin ETB receptor in complex with peptide inverse agonist IRL2500

Crystal structure of human endothelin ETB receptor in complex with peptide inverse agonist IRL2500

Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation

Computational design of thermostabilizing point mutations for G protein-coupled receptors

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