Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10

In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails (“Ala-tails”) that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 RING ligase2)-KLHDC10. Here, we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails and that structural predictions identify candidate Ala-tail-binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail-binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron-recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.