Seminar

Thermostabilization and structure determination of a b1 adrenergic receptor

Dr. María Serrano-Vega

There are 350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of GPCRs and applied it to the b1-adrenergic receptor (bAR). The most stable mutant receptor, bAR-m23, contained six point mutations that led to an apparent Tm 21°C higher than the native protein, and, in the presence of bound antagonist, bAR-m23 was as stable as bovine rhodopsin. In addition, bAR-m23 was significantly more stable in detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand. Inclusion of thermostabilising mutations in a minimal construct with flexible regions removed was successful in producing well-diffracting crystals of bAR using octylthioglucoside. The structure was solved to 2.7 Å resolution. The antagonist cyanopindolol was clearly visible in the ligand binding pocket, and the structure also included bound water molecules, a Na+ ion and ordered detergent molecules.