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State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intr...

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Autores principales: Mitsumoto, Masaya, Sugaya, Kanna, Kazama, Kazuki, Nakano, Ryosuke, Kosugi, Takahiro, Murata, Takeshi, Koga, Nobuyasu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8657880/
https://www.ncbi.nlm.nih.gov/pubmed/34884716
http://dx.doi.org/10.3390/ijms222312906
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author Mitsumoto, Masaya
Sugaya, Kanna
Kazama, Kazuki
Nakano, Ryosuke
Kosugi, Takahiro
Murata, Takeshi
Koga, Nobuyasu
author_facet Mitsumoto, Masaya
Sugaya, Kanna
Kazama, Kazuki
Nakano, Ryosuke
Kosugi, Takahiro
Murata, Takeshi
Koga, Nobuyasu
author_sort Mitsumoto, Masaya
collection PubMed
description G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A(2A) receptor (A(2A)R) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A(2A)R through straight helical connections without any kinks or intervening loops. The chimeric A(2A)R fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.
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spelling pubmed-86578802021-12-10 State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein Mitsumoto, Masaya Sugaya, Kanna Kazama, Kazuki Nakano, Ryosuke Kosugi, Takahiro Murata, Takeshi Koga, Nobuyasu Int J Mol Sci Article G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A(2A) receptor (A(2A)R) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A(2A)R through straight helical connections without any kinks or intervening loops. The chimeric A(2A)R fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs. MDPI 2021-11-29 /pmc/articles/PMC8657880/ /pubmed/34884716 http://dx.doi.org/10.3390/ijms222312906 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Mitsumoto, Masaya
Sugaya, Kanna
Kazama, Kazuki
Nakano, Ryosuke
Kosugi, Takahiro
Murata, Takeshi
Koga, Nobuyasu
State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein
title State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein
title_full State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein
title_fullStr State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein
title_full_unstemmed State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein
title_short State-Targeting Stabilization of Adenosine A(2A) Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein
title_sort state-targeting stabilization of adenosine a(2a) receptor by fusing a custom-made de novo designed α-helical protein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8657880/
https://www.ncbi.nlm.nih.gov/pubmed/34884716
http://dx.doi.org/10.3390/ijms222312906
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