Biochemistry Department, Otago School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand.
Biochem J. 2013 Mar 15;450(3):629-38. doi: 10.1042/BJ20121702.
RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2Ub conjugate to target proteins. In many cases interaction of the E2Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.
E3 连接酶的 RING 结构域促进 Ub（泛素）从 E2Ub 缀合物转移到靶蛋白上。在许多情况下，E2Ub 缀合物与 RING 结构域的相互作用需要其先前的二聚化。我们通过交联实验表明，E2 连接的泛素与 IAP（凋亡抑制剂）蛋白、XIAP（X 连锁 IAP）和 cIAP（细胞 IAP）2 的 RING 同源二聚体界面接触。对 XIAP RING 二聚体的结构和生化分析表明，二聚体界面上的一个芳香族残基是 E2~Ub 结合和 Ub 转移所必需的。芳香族残基的突变会破坏 Ub 转移，但不会破坏与 Ub 的相互作用。这表明亲核攻击硫酯键取决于 Ub 与 RING 结构域之间的精确接触。RING 二聚化是 cIAP 蛋白的关键激活步骤；然而，我们的分析表明，XIAP 的 RING 结构域形成稳定的二聚体，其 E3 连接酶活性不需要激活步骤。
