Fig. 3.

Allosteric conformational changes of ACKR3 activation. (A) Schematic representation of the % differential relative fractional uptake data (apo ACKR3—bound ACKR3) mapped onto the lower part of the AF model of ACKR3 (for clarity, we did not include the C- terminus). Relative fractional uptake is calculated by dividing the experimental uptake (Da) of a peptide by its maximum possible uptake. Scheme depicts reproducible and statistically significant ΔHDX in response to inverse agonists small ligands represented by VUF16840 (VUF25550 giving a similar profile), or agonist small ligand VUF15485. Black regions represent regions with no sequence coverage. Ligand-induced reduction in deuterium uptake is represented in blue while ligand-induced increase in deuterium uptake is in red, according to the scale. (B) Associated deuterium uptake plots showing the relative uptake for peptides from apo or ligand-bound ACKR3 across several deuteration time points and are representative of the intracellular region as indicated at the Top of the plot. Statistically significant changes were determined using Deuteros 2.0 software (48) (P ≤ 0.01): statistically significant time points for the different ligands are represented by a colored star corresponding to the ligand in question (pink, blue, and purple for VUF15485, VUF16840, and VUF25550 respectively). Black stars depict time points that are statistically significant for all three ligands. Uptake plots are the average and SD of 3 technical replicates from the same biological preparation of ACKR3. (C) Calculated ΔlnPF from the MD simulations, mapped on the AF model. (D) Proposed mechanism of activation. The bulky trimethoxybenzamide group of VUF15485 induces a “twist” of the 7TM bundle around the orthosteric pocket, which allosterically triggers TM6 opening on the intracellular side. (E) Probability density distribution of the ionic-lock-residue Cα distances during the MD simulations.