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 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 I196^2.66bF increases thermal stability intrinsically and that mutation S271^4.47bA decreases crystal packing entropy extrinsically, while mutations S193^2.63bC and M233^3.36bC 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.