State-Targeting Stabilization of Adenosine A<sub>2A</sub> 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...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-11-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/23/12906 |
| _version_ | 1797507691299471360 |
|---|---|
| author | Masaya Mitsumoto Kanna Sugaya Kazuki Kazama Ryosuke Nakano Takahiro Kosugi Takeshi Murata Nobuyasu Koga |
| author_facet | Masaya Mitsumoto Kanna Sugaya Kazuki Kazama Ryosuke Nakano Takahiro Kosugi Takeshi Murata Nobuyasu Koga |
| author_sort | Masaya Mitsumoto |
| collection | DOAJ |
| 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<sub>2A</sub> receptor (A<sub>2A</sub>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<sub>2A</sub>R through straight helical connections without any kinks or intervening loops. The chimeric A<sub>2A</sub>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. |
| first_indexed | 2024-03-10T04:53:04Z |
| format | Article |
| id | doaj.art-2ebb7636f5cd406daeb8c29d313f8c70 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T04:53:04Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-2ebb7636f5cd406daeb8c29d313f8c702023-11-23T02:30:09ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-11-0122231290610.3390/ijms222312906State-Targeting Stabilization of Adenosine A<sub>2A</sub> Receptor by Fusing a Custom-Made De Novo Designed α-Helical ProteinMasaya Mitsumoto0Kanna Sugaya1Kazuki Kazama2Ryosuke Nakano3Takahiro Kosugi4Takeshi Murata5Nobuyasu Koga6Department of Structural Molecular Science, School of Physical Sciences, SOKENDAI, The Graduate University for Advanced Studies, Shonan Village, Hayama 240-0193, Kanagawa, JapanDepartment of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, JapanDepartment of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, JapanDepartment of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, JapanDepartment of Structural Molecular Science, School of Physical Sciences, SOKENDAI, The Graduate University for Advanced Studies, Shonan Village, Hayama 240-0193, Kanagawa, JapanDepartment of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, JapanDepartment of Structural Molecular Science, School of Physical Sciences, SOKENDAI, The Graduate University for Advanced Studies, Shonan Village, Hayama 240-0193, Kanagawa, JapanG-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<sub>2A</sub> receptor (A<sub>2A</sub>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<sub>2A</sub>R through straight helical connections without any kinks or intervening loops. The chimeric A<sub>2A</sub>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.https://www.mdpi.com/1422-0067/22/23/12906computational protein designprotein stabilizationde novo designed proteinfusion partner proteinG-protein coupled receptoradenosine A<sub>2A</sub> receptor |
| spellingShingle | Masaya Mitsumoto Kanna Sugaya Kazuki Kazama Ryosuke Nakano Takahiro Kosugi Takeshi Murata Nobuyasu Koga State-Targeting Stabilization of Adenosine A<sub>2A</sub> Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein International Journal of Molecular Sciences computational protein design protein stabilization de novo designed protein fusion partner protein G-protein coupled receptor adenosine A<sub>2A</sub> receptor |
| title | State-Targeting Stabilization of Adenosine A<sub>2A</sub> Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein |
| title_full | State-Targeting Stabilization of Adenosine A<sub>2A</sub> Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein |
| title_fullStr | State-Targeting Stabilization of Adenosine A<sub>2A</sub> Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein |
| title_full_unstemmed | State-Targeting Stabilization of Adenosine A<sub>2A</sub> Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein |
| title_short | State-Targeting Stabilization of Adenosine A<sub>2A</sub> Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein |
| title_sort | state targeting stabilization of adenosine a sub 2a sub receptor by fusing a custom made de novo designed α helical protein |
| topic | computational protein design protein stabilization de novo designed protein fusion partner protein G-protein coupled receptor adenosine A<sub>2A</sub> receptor |
| url | https://www.mdpi.com/1422-0067/22/23/12906 |
| work_keys_str_mv | AT masayamitsumoto statetargetingstabilizationofadenosineasub2asubreceptorbyfusingacustommadedenovodesignedahelicalprotein AT kannasugaya statetargetingstabilizationofadenosineasub2asubreceptorbyfusingacustommadedenovodesignedahelicalprotein AT kazukikazama statetargetingstabilizationofadenosineasub2asubreceptorbyfusingacustommadedenovodesignedahelicalprotein AT ryosukenakano statetargetingstabilizationofadenosineasub2asubreceptorbyfusingacustommadedenovodesignedahelicalprotein AT takahirokosugi statetargetingstabilizationofadenosineasub2asubreceptorbyfusingacustommadedenovodesignedahelicalprotein AT takeshimurata statetargetingstabilizationofadenosineasub2asubreceptorbyfusingacustommadedenovodesignedahelicalprotein AT nobuyasukoga statetargetingstabilizationofadenosineasub2asubreceptorbyfusingacustommadedenovodesignedahelicalprotein |