An Atypical E3 Ligase Module in UBR4 Mediates Destabilization of N-degron Substrates

UBR4 is an E3 ligase (E3) of the N-degron pathway and is involved in neurodevelopment, age-associated muscular atrophy and cancer progression. The location and mechanistic classification of the E3 module within the 600 kDa protein UBR4 remains unknown. Herein, we identify and characterize, at a biochemical and structural level, a distinct E3 module within human UBR4 consisting of a novel “hemiRING” zinc finger, a helical-rich UBR Zinc-finger Interacting (UZI) subdomain, and a predicted backside interacting N-terminal helix. A structure of an E2 conjugating enzyme (E2)-E3 complex provides atomic level insight into the exquisite specificity of the hemiRING towards the E2s UBE2A/B. The UZI subdomain can be considered a component of the E3 module as it has a modest activating effect on the ubiquitin loaded E2 (E2∼Ub), which is complemented by the intrinsically high lysine reactivity of UBE2A. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3 ligase, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains associated with ubiquitin E3 activity.<br/

UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4

UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a ‘hemiRING’ zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2–E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2∼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.</p