There are 20 chemokine receptors that bind their respective chemokines. It is not currently understood how these structurally similar receptors distinguish their ligands; namely, how EC loops and transmembrane domains of these receptors are involved in ligand-binding activities. With the detergent-free GPCRs, we show that it is now possible to design and produce chimeric receptor proteins to study ligand-binding mechanisms. We exchanged the N terminus and 3 EC loops of natural chemokine receptor CXCR4 to append them onto the 7TM α-helices of detergent-free variant CCR5QTY, and systematically studied which ligands it binds. These designer chimeric receptors provide insight into how natural receptors bind their respective ligands. These chimeric receptors with tunable functionality may have applications for bioelectronics sensing devices.
QTY-designed, detergent-free chemokine receptors have been expressed in SF9 insect cells, as well as produced using a low cost and simple E.coli system with much higher throughput. These QTY-designed receptor variants exhibit remarkable heat stability in the presence of arginine additive, retaining ligand binding activity after 100°C treatment. New protein variants can also be designed using the same alpha-helical segments but switching the extracellular (EC) loop, e.g., using variant B’s EC loop to directly attach to variant A’s alpha-helical segments. This approach helps our understanding of the binding mechanism of QTY variants and natural membrane receptors, as well as enables a novel pathway for the design and production of multi-functional, water-soluble membrane receptors, with tunable properties for in vitro and in vivo applications.