A Coiled-Coil-Based Design Strategy for the Thermostabilization of G-Protein-Coupled Receptors

Abstract

The most common methods for generating crystallizable GPCRs are scanning alanine mutagenesis and fusion to crystallization-facilitating partner proteins. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel coiled coil as a domain fold that satisfies both criteria. A selection of antiparallel coiled coils was used for structure-guided substitution of intracellular loop of the {beta}3 adrenergic receptor. Unexpectedly, only the two GPCR variants containing thermostable coiled coils were expressed. We showed that one GPCR chimera is stable upon purification in detergent, retains ligand-binding properties, and can be crystallized. However, the quality of the crystals was not suitable for structure determination. To supply additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop of the antiparallel coiled coil by T4L. Although expression is currently not suitable for structural work, we found that the engineered GPCR is even more stable than the coiled-coil variant. Our approach should be of interest for applications that benefit from stable GPCRs.