Fig. 3: ACKR3-VUN701 β-arrestin2 dependent paratope mapping.
From: Structural basis for selectivity and antagonism in extracellular GPCR-nanobodies
a Model of the ACKR3-VUN701 complex with zoomed view of the orthosteric interactions between VUN701 and ACKR3. Intermolecular interactions are shown as sticks and are colored according to their CDR location. b ACKR3 BRET-based β-arrestin2 assay with increasing CXCL12 (blue) or competition assay of 3.3 nM CXCL12 with increasing WT VUN701 (black), and VUN701 variants D56A (green), K100A (red), and D104A (gray) illustrating the alteration in IC50 due to CDR mutations. N = 3 biologically independent experiments plotted as mean +/- SEM. c) Plot of the change in pKB values for VUN701 CDR alanine mutants (N = 3 biologically independent experiments plotted as mean +/- SEM). Residues with significant alterations in binding marked with asterisks (Ordinary one-way ANOVA). K100A mutation shifted VUN701 binding beyond the limit of detection. d Plot of change in pEC50 values for CXCL12 on orthosteric alanine ACKR3 mutants (N = 3 biologically independent experiments plotted as mean +/- SEM, Ordinary one-way ANOVA). e) Cartoon representation of ACKR3’s orthosteric pocket with residues probed in alanine-scanning marked with circles based on charge (red – negative; blue – positive) or aromaticity (green). f Plot of changes in pKB values for VUN701 on orthosteric alanine ACKR3 mutants (N = 3 biologically independent experiments plotted as mean +/- SEM, Ordinary one-way ANOVA). NDMutant shifted CXCL12’s EC50 beyond accurate determination of VUN701’s IC50; DNEMutant did not express. P < 0.0001 (****); P < 0.031. All receptor residues are numbered in GPCRdb numbering format. Source data are provided as a Source Data file.
