Abstract
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 hydro-phobic 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 di-sulfide-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.
Highlights
Type 2 diabetes is a long-term metabolic disorder that is predicted to affect $10% of the adult population by 2030 (Shaw et al, 2010)
We have previously described that ten thermal-stabilizing mutations were introduced in the transmembrane domain (TMD) structure of glucagon-like peptide-1 receptor (GLP-1R) in complex with two negative allosteric modulators (NAMs), PF-06372222 and NNC0640, and we showed that a disulfide bond (I3175.47bC—G3616.50bC) and a GCGR mimicking mutation C3476.36bF are indispensable for crystallization (Song et al, 2017)
We built a model of GLP-1R based on a previously solved GCGR structure (PDB entry 4l6r; Siu et al, 2013) that we used as a template for mutagenesis design to stabilize the transmembrane bundle, especially the thermodynamic regions revealed in the homologous GCGR structure
Summary
Type 2 diabetes is a long-term metabolic disorder that is predicted to affect $10% of the adult population by 2030 (Shaw et al, 2010). Current treatments of type 2 diabetes include injection of insulin or peptide agonists of glucagon-like peptide-1 receptor (GLP-1R) that provoke the synthesis and release of insulin (Gutniak et al, 1992). The widely accepted twodomain binding model suggests the ECDs of class B receptors recognize the C-terminal helices of their hormone peptide ligands, facilitating binding of the N-terminal region of the peptides to the TMDs for downstream signaling (Hoare, 2005). Discovery of anti-diabetes drugs is limited to peptide agonists including GLP-1, extendin-4 and their analogs (Pabreja et al, 2014), while previously the development of small-molecule drugs to target this receptor was extremely challenging because of the lack of structural information about druggable small-molecule binding sites
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