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A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors

Structure elucidation of inactive-state GPCRs still mostly relies on X-ray crystallography. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel c...

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Autores principales: Amer, Marwa, Leka, Oneda, Jasko, Piotr, Frey, Daniel, Li, Xiaodan, Kammerer, Richard A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10287670/
https://www.ncbi.nlm.nih.gov/pubmed/37349348
http://dx.doi.org/10.1038/s41598-023-36855-1
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author Amer, Marwa
Leka, Oneda
Jasko, Piotr
Frey, Daniel
Li, Xiaodan
Kammerer, Richard A.
author_facet Amer, Marwa
Leka, Oneda
Jasko, Piotr
Frey, Daniel
Li, Xiaodan
Kammerer, Richard A.
author_sort Amer, Marwa
collection PubMed
description Structure elucidation of inactive-state GPCRs still mostly relies on X-ray crystallography. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel coiled coil as a domain fold that satisfies both criteria. A selection of antiparallel coiled coils was used for structure-guided substitution of intracellular loop 3 of the β3 adrenergic receptor. Unexpectedly, only the two GPCR variants containing thermostable coiled coils were expressed. We showed that one GPCR chimera is stable upon purification in detergent, retains ligand-binding properties, and can be crystallized. However, the quality of the crystals was not suitable for structure determination. By using two other examples, 5HTR2C and α2BAR, we demonstrate that our approach is generally suitable for the stabilization of GPCRs. To provide additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop connecting the antiparallel coiled coil by T4L. We found that the engineered GPCR is even more stable than the coiled-coil variant. Negative-staining TEM revealed a homogeneous distribution of particles, indicating that coiled-coil-T4L receptor variants might also be promising candidate proteins for structure elucidation by cryo-EM. Our approach should be of interest for applications that benefit from stable GPCRs.
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spelling pubmed-102876702023-06-24 A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors Amer, Marwa Leka, Oneda Jasko, Piotr Frey, Daniel Li, Xiaodan Kammerer, Richard A. Sci Rep Article Structure elucidation of inactive-state GPCRs still mostly relies on X-ray crystallography. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel coiled coil as a domain fold that satisfies both criteria. A selection of antiparallel coiled coils was used for structure-guided substitution of intracellular loop 3 of the β3 adrenergic receptor. Unexpectedly, only the two GPCR variants containing thermostable coiled coils were expressed. We showed that one GPCR chimera is stable upon purification in detergent, retains ligand-binding properties, and can be crystallized. However, the quality of the crystals was not suitable for structure determination. By using two other examples, 5HTR2C and α2BAR, we demonstrate that our approach is generally suitable for the stabilization of GPCRs. To provide additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop connecting the antiparallel coiled coil by T4L. We found that the engineered GPCR is even more stable than the coiled-coil variant. Negative-staining TEM revealed a homogeneous distribution of particles, indicating that coiled-coil-T4L receptor variants might also be promising candidate proteins for structure elucidation by cryo-EM. Our approach should be of interest for applications that benefit from stable GPCRs. Nature Publishing Group UK 2023-06-22 /pmc/articles/PMC10287670/ /pubmed/37349348 http://dx.doi.org/10.1038/s41598-023-36855-1 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Amer, Marwa
Leka, Oneda
Jasko, Piotr
Frey, Daniel
Li, Xiaodan
Kammerer, Richard A.
A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors
title A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors
title_full A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors
title_fullStr A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors
title_full_unstemmed A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors
title_short A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors
title_sort coiled-coil-based design strategy for the thermostabilization of g-protein-coupled receptors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10287670/
https://www.ncbi.nlm.nih.gov/pubmed/37349348
http://dx.doi.org/10.1038/s41598-023-36855-1
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