An alternative pocket for binding the N‐degrons by the UBR1 and UBR2 ubiquitin E3 ligases

The UBR family of ubiquitin ligases binds to N‐termini of their targets (known as N‐degron) to induce their ubiquitination and degradation via a conserved domain known as UBR‐box. UBR1 and UBR2 share the highest sequence homology among the family, and substantial structural studies were previously performed for substrate binding by the UBR‐boxes of UBR1 and UBR2. Here, we describe a new pocket in the UBR‐boxes of UBR1 and UBR2 for binding the second residues of N‐degrons through determining five co‐crystal structures of the UBR‐boxes with various N‐degron peptides. Together with binding affinities measured by fluorescence polarization, we show that the two highly homologous UBR‐boxes can interact with the second residue of an N‐degron differently. In addition, the UBR‐boxes undergo different conformational changes when binding N‐degrons. Furthermore, we demonstrate that the sidechain of the third amino acid of an N‐degron has no contribution to binding the UBR‐boxes. These findings represent a new conceptual advancement for the UBR E3 ligases and the new insights described here can be leveraged for developing their selective ligands for research and potential therapies.