The backside β-turn is a key structural element of Rad6-family E2 ubiquitin-conjugating enzymes.

Abstract

Protein ubiquitination regulates diverse cellular processes, and its dysregulation contributes to human disease, including cancer. E2 ubiquitin‑conjugating enzymes share a conserved UBC fold in which surface loops fine‑tune catalysis and partner interactions, yet the roles of individual loops remain incompletely defined. Here, we identify loop 3-a component of the "backside" β2-β3 hairpin-as a conserved structural and allosteric element in Rad6‑family E2s. Structural and bioinformatic analyses of yeast Rad6 and its human homologs (UBE2A/UBE2B) reveal that loop 3 forms an overlapping triple β‑turns, with variable first turn and a highly conserved second/third turn that links catalytic regulation to E3 ligase engagement. Systematic mutagenesis of the yeast Rad6 backside β‑turn (residues 42-51) shows that this element is required in vivo for Bre1‑dependent histone H2B Lys123 monoubiquitination, Rad18‑dependent PCNA monoubiquitination, and Ubr1/Ubr2‑dependent polyubiquitination and degradation of Sml1 and N‑end rule substrates, and related biological processes. Charge‑reversal mutations at backside β‑turn Glu49 and Asp50 disrupt E3 binding, whereas cancer‑relevant substitutions in kink‑inducing prolines (Pro43/Pro47) impair mono‑ and polyubiquitination without abolishing E3 interactions. Certain backside β‑turn mutations, including cancer-relevant variants, compromise steady-state levels following DNA damage, revealing them as conditional null or loss-of-function alleles. NMR spectroscopy demonstrates that Pro43/Pro47 mutations induce long‑range structural perturbations from backside β‑turn into the front‑face catalytic pocket, correlating with reduced in vitro ubiquitination activity. Deletion or alanine replacement of the β‑turn destabilizes yeast Rad6 and human UBE2A/UBE2B. Together, these findings establish the loop 3/backside β‑turn as a critical structural element of Rad6‑family enzymes.