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.

UBR4 是 N-降解决定子途径的 574 kDa E3 连接酶 (E3)，在神经发育、年龄相关性肌肉萎缩和癌症中发挥作用。进行泛素（Ub）转移的催化模块仍然未知。在这里，我们鉴定并表征了人类 UBR4 中的一个独特的 E3 模块，该模块由“hemiRING”锌指、富含螺旋的 UBR 锌指相互作用 (UZI) 子结构域和可作为亲和因子的 N 端区域组成。 E2结合酶(E2)。 E2-E3 复合物的结构提供了原子水平的洞察，以了解 hemiRING 对同源 E2 UBE2A/UBE2B 的特异性决定因素。通过变构机制，UZI 子结构域适度激活 Ub 负载的 E2 (E2 ∼ Ub)。我们建议 UBE2A 固有的高赖氨酸反应性补充了减弱的激活，并且它们的合作赋予了对底物特异性和最佳降解动力学重要的反应性特征。这些发现揭示了神经元 N-degron E3 的机制基础、UBE2A 的特异性募集，并强调了具有 Ub E3 活性的交叉支撑结构域的结构多样性未被充分认识。