Fig. 4: UBE3A mutations demarcate a ubiquitin-binding exosite.

a Crystal structure (PDB: 1C4Z) of the HECT domain of UBE3A (gray) bound to UBCH7 (gold). Mutations identified in our screen found in the catalytic C-lobe (purple), the E2 binding interface (blue), and two uncharacterized regions (pink and green) are shown. All hyperactivating mutations are shown in red. b Crystal structure of the HECT domain of NEDD4 (gray) bound to a monomeric ubiquitin molecule (pink) at its exosite (PDB: 4BBN). Note the binding of ubiquitin to NEDD4 occurs at a site homologous to a cluster of mutations (pink) in UBE3A. c, d Surface (left) and ribbon (right) representations of the ubiquitin-binding interface for UBE3A (c) and NEDD4 (d). Ubiquitin is represented in pink. Dotted boxes represent the position of hydrophobic binding pocket in NEDD4 that accepts L73 of the ubiquitin C-terminus. Surface charges are indicated by color: positive (blue), negative (red), uncharged (white). e, f Rosetta simulations showing ensembles of low energy (high confidence) conformations for the C-terminus of ubiquitin docked to UBE3A. The divergent orientation of the ubiquitin tail caused by the Q588E mutation in UBE3A is noted by the arrow. g, h Interface score versus RMSD plots showing Rosetta simulations under 880 Rosetta Energy Units (REU). The positions of the low energy models are noted as stars according to the color scheme used in e, f. The gray dotted box demarcates WT-like tail conformations whereas the black dotted box indicates the Q588E tail conformation.