The MLLE Domain of the Ubiquitin Ligase UBR5 Binds to Its Catalytic Domain to Regulate Substrate Binding

Abstract E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2 conjugating enzyme to a substrate. UBR5, a HECT-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein-protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C-terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein-protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.