Fig. 1

Peptide binding of the USP8 MIT domain. (A) Schematic representation of USP8 domain organization: N-terminal MIT domain (MIT), rhodanese-like domain (Rhod), WW-like domain (WW-like), C-terminal USP catalytic domain (USP). (B) USP8 MIT peptide-ligands identified through ProP-PD selections. The percent of NGS counts associated with each peptide is indicated. The MIM1-like motif found in four peptides is highlighted in bold, and motif is indicated to the right for comparison. (C) SPOT array alanine scanning of the Girdin_{1192 − 1207} peptide binding to the MIT domain. Amino acid residues disrupting binding when mutated to alanine are shown in bold. Signal intensities were normalized to the wild-type (Wt) and displayed as average percent signal change; Nc: negative control (scrambled sequence). (D) AF3 models of superimposed MIM1-like motif containing peptides binding to the USP8 MIT domain. The peptide sequence alignment depicts motif residues in bold on grey background, and details of the docking of the key motif positions are shown by insets to the right (1) and left (4 and 8). (E) AF3 model of the Girdin_{1192 − 1207} peptide (cyan) binding to the MIT domain. USP8 MIT amino acid residues interacting with the peptide are shown in blue. (F) SPOT array alanine scanning of DBP_{116 − 131} peptide binding to the MIT domain. Amino acid residues disrupting binding when mutated to alanine (or glycine in case of wild type alanine) are shown in bold. Signal intensities were treated as describe for panel (C). (G) AF3 model of DBP_{116 − 131} peptide (orange) binding to the MIT domain. USP8 MIT domain amino acid residues interacting with the peptide are shown in blue. (H) AF3 superimposed models of Girdin_{1192 − 1207} peptide (cyan) and DBP_{116 − 131} peptide (orange) binding to the same binding pocket as the MIM1-like containing peptides in (D)