Fig. 3. Ligand binding kinetics of truncated ACKR3 variants.

(A) Representative example of SDS-PAGE of purified bril-ACKR3_WT, bril-ACKR3_d17 and bril-ACKR3_d29. Addition of PNGase F deglycosylates the receptor. (B) Representative example of thermal unfolding of ACKR3 variants in complex with CCX662 and CXCL12_LRHQ measured using CPM fluorescence (32). (C) Representative example of CXCL12_WT association to ACKR3 variants at 5 nM chemokine detected by flow cytometry. (D) Percent of the association curves corresponding to the slower phase of chemokine association determined from fitting association curves at 10 nM CXCL12_WT to a two-phase exponential equation. Bars represent the average and standard errors of three or more experiments. ACKR3_d29 has a larger slow component than ACKR3_WT (P<0.05 as determined from one-way anova with Dunnett’s multiple comparison test). (E) Mean and standard errors from three or more measurements of k_obs values for the slow phase in (D). (F) Representative example of arrestin recruitment to ACKR3 variants determined from BRET experiments after addition of 10 nM CXCL12_WT. (G) Average and standard errors of k_obs values from fitting three time-resolved BRET experiments to single exponential equations. (H) Representative example of CXCL12_WT dissociation from ACKR3:CXCL12 complexes in Sf9 cells. (I) Mean and standard errors of CXCL12_WT dissociative half-life determined from fitting three or more dissociation curves to a single-phase exponential equation. The dissociative half-life of bril-ACKR3_d29 is significantly shorter than ACKR3_WT (P<0.001 as determined from one-way anova with Dunnett’s multiple comparison test). (J) Schematic representation of CXCL12 binding equilibria for ACKR3_WT and ACKR3_d29 highlighting the faster dissociation rate but unchanged association rate of CXCL12 binding to the truncated receptor.