Abstract
Structure elucidation of inactive-state GPCRs still mostly relies on X-ray crystallography. 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 3 of the β3 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. By using two other examples, 5HTR2C and α2BAR, we demonstrate that our approach is generally suitable for the stabilization of GPCRs. To provide additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop connecting the antiparallel coiled coil by T4L. We found that the engineered GPCR is even more stable than the coiled-coil variant. Negative-staining TEM revealed a homogeneous distribution of particles, indicating that coiled-coil-T4L receptor variants might also be promising candidate proteins for structure elucidation by cryo-EM. Our approach should be of interest for applications that benefit from stable GPCRs.