Fig. 1: Binding affinities and structures of arrestin2 in complex with four different phosphobarcodes.

a Sequence representation of V2Rpp-FP, V2Rpp-1, V2Rpp-3, V2Rpp-4, and V2Rpp-6-7. According to a previously published structure, phospho-residues that were expected to bind to arrestin2 are marked blue. The corresponding dissociation constant (Kd value) of each peptide and melting temperature shift (ΔTm value) caused by Fab 30 incubation are shown in the right panels and were determined by Cou-facilitated FRET assay and differential scanning fluorimetry (DSF), respectively. See Supplementary Fig. 2 in the supplementary file. b A current model of the phospho-barcoding interaction with arrestin2 according to solved crystal structures or Cryo-EM structures of arrestin2 complexes. The orange balls of V1 to V7 indicate the phospho-binding pockets of arrestin found in the arrestin2-V2Rpp-FP complex (PDB: 4JQI). The blue balls β1–β7 indicate that a phosphate or a negatively charged residue interacts with the specific sites of arrestin, that are determined from the arrestin2-V2T complex (6NI2) and arrestin2-β1V2R6P complex (PDB: 6TKO). c–f The 2Fo-Fc annealing omits electron density maps of V2Rpp-1, V2Rpp-3, V2Rpp-4, and V2Rpp-6-7. All maps were contoured at 1.0 σ. The orange balls indicate that the phosphorylated peptide remains bound to arrestin, as observed in the previously solved arrestin2-V2Rpp-FP complex structure, whereas the green balls indicate loss of the original interaction of the phospho-residue. The purple ball represents the found phospho-binding pocket of arrestin in the arrestin2-V2Rpp-4 complex structure. g The specific phospho-binding patterns of arrestin revealed from V2Rpp/arrestin2 complexes. The blue buttons indicate the binding of the selective V2R phospho-peptide in accordance with the predicted phospho-pattern according to the previously solved arrestin2-V2Rpp-FP complex structure. The purple button suggests a discovered phospho-binding pocket of arrestin2 that interacts with V2Rpp-4. Gray buttons indicate that no phosphate interaction was found at the corresponding phospho-binding pockets of arrestin.