Experimental design of the smFRET system. A) A single receptor molecule (blue) was labeled with donor (D) and acceptor (A) fluorophores, inserted into a phospholipid (yellow) nanodisc (green), and immobilized on a quartz slide via biotin (brown circle)-neutravidin (grey rectangle) attachment. A prism facilitates total internal reflection of the excitation laser to excite only donor fluorophores close to the surface. B) Cartoon depicting inactive (left) and active (right) receptor conformations. C) Two representative single-molecule time traces for apo-ACKR3. In both examples, the donor (green) and acceptor (red) intensities are shown in the top panel and the corresponding FRET efficiency (black) is shown in the bottom panel.

ACKR3 exhibits greater conformational flexibility compared to CXCR4. A) FRET efficiency histogram of apo-CXCR4 (left, black trace) resolved into three distinct conformational states: a high-FRET state corresponding to the inactive receptor conformation (R, blue), a low-FRET active receptor conformation (R*, red) and an intermediate conformation (R’, gray). The fractional populations of each state obtained from global analysis are indicated. The receptor is mostly in the inactive conformation. A transition density probability (TDP) plot (right) displays the relative probabilities of transitions from an initial FRET state (x-axis) to a final FRET state (y-axis). For apo- CXCR4, transitions between R and R’ states are observed most frequently. B) Addition of CXCL12 to CXCR4 shifted the conformational distribution to the low-FRET R* state and resulted in more transitions between all three FRET states. C) The broad FRET efficiency histogram of apo-ACKR3 (left, black trace) is resolved into four distinct conformational states: inactive R (blue), active R* (red), inactive-like R’ (light blue), and active-like R*’ (pink). Little conformational preference is observed among these states. Moreover, all possible sequential state-to-state transitions are observed (right). D) Addition of CXCL12 to ACKR3 shifted the conformational distribution to the low-FRET R* state, which was also reflected in the transition probabilities. In all cases, data sets represent the analysis of at least three independent experiments.

A small molecule inhibitor shifts the ACKR3 conformational population to the inactive FRET state, while CXCR4 is largely unaffected. A) FRET distributions and TDP of apo-CXCR4 repeated from Fig. 2A for comparison. B) Treatment of CXCR4 with the inhibitor IT1t had little impact on the FRET distribution (compared to 2A), but increased transition probabilities compared to the apo-receptor. C) FRET distributions and TDP of apo-ACKR3 repeated from Fig. 2C for comparison. D) Treatment of ACKR3 with VUF16480, an inverse agonist, shifted the conformational distribution and TDPs to the high-FRET inactive R conformation. Data sets represent the analysis of at least three independent experiments. The overall FRET efficiency envelopes for the samples are represented by the black traces.

CXCL12 variants containing mutations to the N-terminus promoted active receptor conformations in both CXCR4 and ACKR3. A) FRET distributions and TDP for apo-CXCR4 repeated from Fig. 2A for reference. B) Addition of CXCL12P2G to CXCR4 promoted a shift to the low-FRET active (R*) conformation and an increase in state-to- state transition probabilities. C) CXCL12LRHQ led to a more subtle shift to the R* conformation of CXCR4 without affecting the transition probabilities. D) FRET distributions and TDPs for apo-ACKR3 repeated from Fig. 2C. E) Treatment of ACKR3 with CXCL12P2G displayed a shift to low-FRET, R*’ and R* states, while reducing the transition probabilities for R’ ↔ R*’ and R*’ ↔ R* transitions relative to the apo-receptor. F) CXCL12LRHQ treatment of ACKR3 shifted the FRET distribution to the low-FRET R* active state and promoted R*’ ↔ R* transitions relative to the apo-receptor. In all cases, the data sets represent the analysis of at least three independent experiments. The overall FRET efficiency envelopes for the samples are represented by the black traces.

Replacement of Y2576.40 with the corresponding residue in CXCR4 (leucine) reduces conformational heterogeneity of ACKR3. A) Structure of ACKR3 bound with CXCL12WT (PDBID: 7SK3) highlighting the location of Y2576.40 (purple) (16). B) FRET efficiency distributions and TDP of WT ACKR3 in the apo-state, repeated from Fig. 2C. C) The mutation Y2576.40L shifted the conformational landscape of the apo-receptor to the high-FRET inactive R conformation at the expense of active R* and active-like R*’ conformations, and also reduced the probability of state-to-state transitions. D) FRET efficiency distributions and TDP of WT ACKR3 treated with CXCL12, repeated from Fig. 2D. E) Treatment of Y2576.40L ACKR3 with CXCL12 promoted more low-FRET active R* and active-like R*’ states. Data sets represent the analysis of at least three independent experiments. The overall FRET efficiency envelopes for the samples are represented by the black traces.

Schematic of the conformational energy landscapes for CXCR4 and ACKR3, highlighting the differences in the responsiveness of the two receptors to ligands. CXCR4 populates three distinct conformations, shown here as wells on the energy landscape. Apo-CXCR4 is predominantly in the inactive R state. The receptor is converted incompletely to R* with CXCL12WT treatment, while the antagonist IT1t has little impact on the conformational distribution. Though CXCL12P2G is an antagonist, the ligand promoted a detectable shift to the active R* state, suggesting TM6 movement is not sufficient for CXCR4 activation. In contrast, ACKR3 populates four distinct conformations and shows little preference among them in the apo-form. The inverse agonist, VUF16840, shifts the population to the inactive R conformation, while the agonists CXCL12WT and CXCL12P2G promote the R* and R*’ populations of ACKR3. Despite stabilizing different levels of the active R* state and active-like intermediate R*’ state, both CXCL12WT and CXCL12P2G are agonists of ACKR3. The flexibility of ACKR3 may contribute to the ligand-promiscuity of this atypical receptor. Figure created with biorender.com

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