Structural Visualization of HECT-E3 Ufd4 accepting and transferring Ubiquitin to Form K29/K48-branched Polyubiquitination on N-degron

Proteins with destabilizing N-terminal residues are degraded via K48-linked ubiquitination-dependent N-degron pathway, during which the HECT-type E3 ligase Ufd4 can further augment polyubiquitination events on these proteins to accelerate their degradation. How Ufd4 increase polyubiquitination on ubiquitinated N-degrons remains unclear. Here, we biochemically determined that Ufd4 preferentially ubiquitinates the proximal ubiquitin in K48-linked ubiquitin chain in K29-linkage specific manner and revealed high-resolution cryo-EM structures of Ufd4 accepting (E2-to-E3 state, 3.52 Å) and transferring ubiquitin-thioester (E3-to-substrate state, 3.31 Å), respectively. The N-terminal ARM region and HECT domain C-lobe of Ufd4 were identified and characterized as key structural elements that together recruit K48-linked diUb and orient Lys29 of its proximal Ub to the active cysteine of Ufd4 for K29-linked branched ubiquitination. These structures not only provide mechanistic insights into the architecture of the Ufd4 complex but also show the full picture of the Ub transferring cascades of HECT-type E3 ligase.