Fig. 4: The inhibitor binding pocket is unique among NEDD4 family members.
From: Structure-based design of potent and selective inhibitors of the HECT ligase NEDD4
A Amino acids alignment, based on 3D structures, of NEDD4 and NEDD4 family members indicating the conserved and variable residues of the inhibitor binding pocket. Cys627, covalently bound to the inhibitor is highlighted in yellow, Phe residues corresponding to amino acid 553 in NEDD4 are highlighted in orange, Leu residues corresponding to the same position are in red. B Close-up view of the inhibitor binding pocket of ITCH (PDB 3TUG). Residue Cys610, positioned differently within the pocket, is highlighted. C Ub-TR-FRET assay performed using the HECT of NEDD4 (left panel) or NEDD4-Like (right panel) in absence or presence of compound 15 at the indicated concentrations. Plots show time-point measurements of FRET signal to background ratio (% S/B) as function of time (minutes). Mean ± SEM (n = 2). D Superposition of the N-lobes of HECT NEDD4 in complex with compound 15 (PDB 9H9T) and HECT NEDD4-Like structures (PDB 2ONI). The presence of Phe608 (orange) in NEDD4-Like hinders inhibitor binding. Leu residue corresponding to the same position in NEDD4 is in green. The respective Cys residues are highlighted using the same colour scheme. E Ub chain formation assay with the indicated HECT domains treated or not with compound 15 at 10 µM concentration. Reactions were stopped at 30 min by quenching the reaction with Laemmli buffer with reducing agent. IB as indicated. Mono-, di- and polyUb chains are labeled on the right. Bottom, Coomassie staining showing comparable loading of proteins. F Schematic representation of the target validation workflow using a mass spectrometry-based approach. A549 wild-type (WT) and NEDD4 knockout (KO) cell lines were treated with 1 µM compound 17B for 4 h. A total of 2 mg of urea lysates were subjected to streptavidin-based immunoprecipitation. The bound proteins were eluted, separated by gel electrophoresis, and analysed by LC-MS/MS. G One-tenth of the protein complexes bound to streptavidin beads were analysed by IB, as indicated. For input controls, 30 µg of total cell lysates were loaded.
