Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Nature. 2011 Jun 2;474(7349):105-8. doi: 10.1038/nature09966. Epub 2011 May 1.
Although the functional interaction between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signalling, the criteria that define an active E2-E3 pair are not well established. The human E2 UBCH7 (also known as UBE2L3) shows broad specificity for HECT-type E3s, but often fails to function with RING E3s in vitro despite forming specific complexes. Structural comparisons of inactive UBCH7-RING complexes with active UBCH5-RING complexes reveal no defining differences, highlighting a gap in our understanding of Ub transfer. Here we show that, unlike many E2s that transfer Ub with RINGs, UBCH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UBCH7 exhibits activity with the RING-in-between-RING (RBR) family of E3s that includes parkin (also known as PARK2) and human homologue of ariadne (HHARI; also known as ARIH1). Found in all eukaryotes, RBRs regulate processes such as translation and immune signalling. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn(2+)-binding domains, in-between-RING (IBR) and RING2 domains, which together define this E3 family. We show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ∼Ub), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UBCH7, an E2 involved in cell proliferation and immune function, and indicate a novel mechanism for an entire class of E3s.
虽然泛素连接酶（E2s）和泛素连接酶（E3s）之间的功能相互作用对于泛素（Ub）信号转导至关重要，但定义活性 E2-E3 对的标准尚未确定。人类 E2 UBCH7（也称为 UBE2L3）对 HECT 型 E3 具有广泛的特异性，但尽管形成了特异性复合物，在体外通常无法与 RING E3 一起发挥作用。无活性 UBCH7-RING 复合物与活性 UBCH5-RING 复合物的结构比较没有显示出明确的差异，这突显了我们对 Ub 转移理解的差距。在这里，我们表明，与许多与 RING 一起转移 Ub 的 E2 不同，UBCH7 缺乏与赖氨酸的内在、E3 独立反应性，解释了它对 HECT 的偏好。尽管缺乏赖氨酸反应性，UBCH7 仍具有与包括 parkin（也称为 PARK2）和人类 ariadne（HHARI；也称为 ARIH1）同源物在内的 RING-in-between-RING（RBR）家族 E3 的活性。RBR 家族存在于所有真核生物中，调节翻译和免疫信号等过程。RBRs 包含一个典型的 C3HC4 型 RING，其后是两个保守的 Cys/His 丰富的 Zn（2+）结合结构域，在 RING 之间（IBR）和 RING2 结构域之间，这一起定义了这个 E3 家族。我们表明，RBRs 像 RING/HECT 杂种一样发挥作用：它们通过 RING 结构域结合 E2，但通过必需的硫酯键连接的 Ub（表示为 ∼Ub）转移 Ub，这需要 RING2 中的保守半胱氨酸残基。我们的结果定义了 UBCH7 的 E3 功能框架，UBCH7 是一种参与细胞增殖和免疫功能的 E2，并表明了一类全新的 E3 机制。
