Nakasone Mark A, Buetow Lori, Gabrielsen Mads, Ahmed Syed F, Majorek Karolina A, Sibbet Gary J, Smith Brian O, Huang Danny T
Cancer Research UK Scotland Institute, Glasgow, UK
Cancer Research UK Scotland Institute, Glasgow, UK.
Life Sci Alliance. 2025 Aug 5;8(10). doi: 10.26508/lsa.202503394. Print 2025 Oct.
RING family ubiquitin ligases (E3s) employ the RING domain to recruit the E2 thioester ubiquitin (E2∼Ub) intermediate to catalyze the transfer of ubiquitin (Ub) to substrates. A cationic Arg linchpin (LP) residue in the RING domain plays a key role in stabilizing the interface with E2∼Ub, but the identity of the LP residue varies across E3s. Here, we investigate how the LP residue contributes to ubiquitination. Using the model RNF38 system, we demonstrate that substitution of LP to the other 19 available amino acids modulates ubiquitination, ranging from minor reduction to complete abolition. The identity of the LP residue influences E2∼Ub binding but does not correlate with E3 activity. NMR and X-ray crystallography analyses reveal that RNF38 LP variants stabilize E2∼Ub in a catalytically competent conformation to varying degrees. By altering the LP residue in XIAP, we show that the XIAP variant promotes E2∼Ub stabilization and enhances substrate ubiquitination in cells. Our work demonstrates the importance of the LP residue in modulating E2∼Ub conformation to control ubiquitination.
RING家族泛素连接酶（E3s）利用RING结构域招募E2硫酯泛素（E2∼Ub）中间体，以催化泛素（Ub）向底物的转移。RING结构域中的阳离子精氨酸关键残基（LP）在稳定与E2∼Ub的界面中起关键作用，但LP残基的身份在不同的E3s中有所不同。在这里，我们研究LP残基如何促进泛素化。使用模型RNF38系统，我们证明将LP替换为其他19种可用氨基酸会调节泛素化，范围从轻微减少到完全消除。LP残基的身份影响E2∼Ub结合，但与E3活性无关。核磁共振（NMR）和X射线晶体学分析表明，RNF38 LP变体在不同程度上以催化活性构象稳定E2∼Ub。通过改变XIAP中的LP残基，我们表明XIAP变体促进E2∼Ub稳定并增强细胞中的底物泛素化。我们的工作证明了LP残基在调节E2∼Ub构象以控制泛素化中的重要性。
