Mutagenesis facilitated crystallization of GLP-1R
| Autor: | Yueming Xu, Yang Wang, Gaojie Song, Deqiang Yao, Yao Peng, Kaiwen Liu, Houchao Tao, Yuxia Wang, Haiguang Liu |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
crystallization
Allosteric regulation G-protein-coupled receptors membrane proteins Computational biology Peptide hormone Biochemistry law.invention 03 medical and health sciences 0302 clinical medicine law General Materials Science Crystallization Receptor lcsh:Science 030304 developmental biology G protein-coupled receptor 0303 health sciences glucagon-like peptide-1 receptor Chemistry General Chemistry Condensed Matter Physics mutations Research Letters molecular dynamic simulations Transmembrane domain Membrane protein lcsh:Q Signal transduction 030217 neurology & neurosurgery |
| Zdroj: | IUCrJ IUCrJ, Vol 6, Iss 6, Pp 996-1006 (2019) |
| ISSN: | 2052-2525 |
| Popis: | The ∼100 mutations used during the structural determination of GLP-1R suggest multiple mutagenesis strategies, which may find applications in the crystallization of G-protein-coupled receptors and other membrane proteins, and potentially in functional and pharmacological studies by locking proteins into specific conformations. 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. |
| Databáze: | OpenAIRE |
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