Hejeong Shin

PROTEOLYSIS1 (PRT1), an N-recognin of Arabidopsis thaliana, recognizes the N-terminal aromatic hydrophobic residue (Tyr/Phe/Trp) of its substrates and ubiquitylates them for degradation by the ubiquitin-proteasome system. Herein, we report the structures of the ZZ domain of PRT1 (PRT1^ZZ) in complex with bulky hydrophobic N-degron peptides. Unlike other ZZ domains, PRT1^ZZ has an unusual binding site with two hydrophobic regions. The N-terminal aromatic residues of N-degrons interact with Ile333 and Phe352 in the flexible loops, which undergo a conformational change. Notably, we identify a third residue from the N-terminus of the substrate that participates in the hydrophobic network with PRT1^ZZ. Moreover, AlphaFold prediction and biochemical assays revealed that the tandem RING1 and RING2 domains of PRT1 interact intramolecularly. The dimeric RING domains in a single protein represent a unique feature among the RING-type E3 ligases. The biochemical assays using the N-terminal tyrosine-exposed substrate, BIG BROTHER, show that the intramolecular RING dimer is essential for PRT1’s robust activity. Therefore, this study expands our knowledge of the structural repertoire in the N-degron pathway and provides insights into the regulation of E3 ligases containing tandem RING domains.

PROTEOLYSIS1 (PRT1), an N -recognin of Arabidopsis thaliana , has a specificity for recognizing the N -terminal aromatic hydrophobic residue (Tyr/Phe/Trp) of its substrates, subsequently degrading them through ubiquitylation. Here, we report the complex structures of the ZZ domain of A. thaliana PRT1 (PRT1^ZZ) with bulky hydrophobic N -degron peptides. Unlike other ZZ domains, the binding site of PRT1^ZZ has a novel structure organized into two hydrophobic regions. The N -terminal aromatic residues of N -degron interact hydrophobically with Ile333 and Phe352 in the flexible loops, which undergo dramatic conformational change. Notably, we also identified a third N -degron residue that participates in the hydrophobic network with N -recognin. Moreover, the ubiquitylation assay of PRT1 using the N -terminal tyrosine-exposed substrate BIG BROTHER showed that the tandem RING organization in PRT1 is critical for its robust activity. Therefore, the current study expands our knowledge of the structural repertoire in the N -degron pathway and provides insights into the regulation of E3 ubiquitin ligases containing tandem RING domains.