Merkley Nadine, Shaw Gary S
Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
J Biol Chem. 2004 Nov 5;279(45):47139-47. doi: 10.1074/jbc.M409576200. Epub 2004 Aug 24.
E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.
E2 缀合酶与泛素形成硫酯中间体，随后该中间体被转移至靶向降解的底物蛋白上。虽然所有 E2 蛋白都包含一个形成硫酯的催化结构域，但有几种 E2（II 类）具有 C 末端延伸，据推测可控制底物识别、二聚化或多聚泛素链形成。在此，我们展示了来自酿酒酵母的 II 类 E2 缀合酶 Ubc1 的新型溶液结构。该结构表明，N 末端催化结构域采用了其他 E2 蛋白典型的α/β折叠。这个结构域通过一个 22 个残基的柔性连接子与其 C 末端结构域在物理上分隔开。C 末端结构域采用了一个三螺旋束，我们已将其鉴定为泛素相关结构域（UBA）。核磁共振化学位移扰动实验表明，这个 UBA 结构域以区域选择性方式与泛素相互作用。Ubc1 的这种双结构域结构被用于鉴定其他含有 UBA 的 II 类 E2 蛋白，包括人类 E2-25K，它们可能具有相似的结构，并用于确定 UBA 结构域在促进多聚泛素链形成中的作用。
