Conformational Dynamics Of Classical And Atypical Gpcrs, Cxcr4 And Ackr3, By Single-Molecule Fret

The G protein-coupled receptors (GPCRs) CXCR4 and ACKR3 both bind the same natural agonist CXCL12, yet CXCR4 signals through G proteins and arrestins, while ACKR3 only couples to the latter. This naturally occurring receptor-mediated signaling bias also manifests in mutations to CXCL12 that cause the agonist to no longer activate CXCR4, but still acts as an ACKR3 agonist. How does CXCL12 binding lead to the different activity of these related receptors? Here, we have investigated the conformational dynamics of the two receptors by single-molecule Förster resonance energy transfer (smFRET) using probes to detect translocation of transmembrane helix 6 (TM6) of purified receptors reconstituted in nanodiscs. Our results show a consistent picture of both CXCR4 and ACKR3 occupying multiple conformational states in the absence of ligands, which collapse to mostly a single FRET state with the addition of agonist. Additionally, the system responds as expected to antagonists and mutants of CXCL12 induce opposite conformational changes for CXCR4 and ACKR3. Additionally, our experimental setup is amenable to the addition of ancillary binders, such as G proteins and arrestins, and we observe further population shifts when these are included. In this way we are able to observe differences in activation of ACKR3 and CXCR4 by the same ligand. The optimization of our single-molecule system and investigations into the activation differences of CXCR4 and ACKR3 will be discussed along with potential caveats.