G protein-coupled receptors (GPCRs) form oligomers that are viable drug targets for the treatment of several diseases including Parkinson’s and schizophrenia. However, design of structurally inspired pharmaceuticals to target specific oligomers has been hindered by experimental difficulties including obtaining sufficient quantities of functional protein for structural characterization. To address these issues, we have developed an expression system in yeast to produce and purify milligram quantities of the full length wild type adenosine A2a receptor (A2aR) and several mutant A2aR variants for oligomer separation by size exclusion chromatography and biophysical study. Additionally, we have taken an integrated approach to combine thermodynamic modeling and high throughput screening to engineer an A2aR construct void of free cysteines that is properly folded. This construct was sought to facilitate the application of site-specific techniques such as electron paramagnetic resonance (EPR) that present a unique opportunity to probe structural dynamics to link structure and function of A2aR homo-dimers without requiring large quantities of protein. To this end, a combinatorial mutant library was designed for site saturation mutagenesis at all six native transmembrane cysteines and subsequently screened for expression, membrane localization and ligand activity with a flow activated cell sorting (FACS) based fluorescent ligand binding assay. Successful variants were identified by sequence verification and modeled to predict thermodynamically stable combinations.  This work represents a robust approach which could be extended to other GPCR oligomers to elucidate crucial structural information, including the identification of the oligomer interface.

Engineering a GPCR for biophysical analysis of oligomer interactions

253rd National Meeting (2017)

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