Mutagenesis facilitated crystallization of GLP-1R
The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs availa...
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doaj-dfc11f5493104f83aa57e261a0b1bcd72020-11-25T01:07:38ZengInternational Union of CrystallographyIUCrJ2052-25252019-11-0166996100610.1107/S2052252519013496tj5027Mutagenesis facilitated crystallization of GLP-1RYueming Xu0Yuxia Wang1Yang Wang2Kaiwen Liu3Yao Peng4Deqiang Yao5Houchao Tao6Haiguang Liu7Gaojie Song8Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, People's Republic of ChinaiHuman Institute, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, People's Republic of ChinaComplex Systems Division, Beijing Computational Science Research Center, Beijing 100193, People's Republic of ChinaiHuman Institute, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, People's Republic of ChinaiHuman Institute, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, People's Republic of ChinaiHuman Institute, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, People's Republic of ChinaiHuman Institute, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, People's Republic of ChinaComplex Systems Division, Beijing Computational Science Research Center, Beijing 100193, People's Republic of ChinaShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, People's Republic of ChinaThe class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins.http://scripts.iucr.org/cgi-bin/paper?S2052252519013496mutationsg-protein-coupled receptorsglucagon-like peptide-1 receptormembrane proteinsmolecular dynamic simulationscrystallization |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yueming Xu Yuxia Wang Yang Wang Kaiwen Liu Yao Peng Deqiang Yao Houchao Tao Haiguang Liu Gaojie Song |
spellingShingle |
Yueming Xu Yuxia Wang Yang Wang Kaiwen Liu Yao Peng Deqiang Yao Houchao Tao Haiguang Liu Gaojie Song Mutagenesis facilitated crystallization of GLP-1R IUCrJ mutations g-protein-coupled receptors glucagon-like peptide-1 receptor membrane proteins molecular dynamic simulations crystallization |
author_facet |
Yueming Xu Yuxia Wang Yang Wang Kaiwen Liu Yao Peng Deqiang Yao Houchao Tao Haiguang Liu Gaojie Song |
author_sort |
Yueming Xu |
title |
Mutagenesis facilitated crystallization of GLP-1R |
title_short |
Mutagenesis facilitated crystallization of GLP-1R |
title_full |
Mutagenesis facilitated crystallization of GLP-1R |
title_fullStr |
Mutagenesis facilitated crystallization of GLP-1R |
title_full_unstemmed |
Mutagenesis facilitated crystallization of GLP-1R |
title_sort |
mutagenesis facilitated crystallization of glp-1r |
publisher |
International Union of Crystallography |
series |
IUCrJ |
issn |
2052-2525 |
publishDate |
2019-11-01 |
description |
The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins. |
topic |
mutations g-protein-coupled receptors glucagon-like peptide-1 receptor membrane proteins molecular dynamic simulations crystallization |
url |
http://scripts.iucr.org/cgi-bin/paper?S2052252519013496 |
work_keys_str_mv |
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