Wang Min, Cheng Dongmei, Peng Junmin, Pickart Cecile M
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
EMBO J. 2006 Apr 19;25(8):1710-9. doi: 10.1038/sj.emboj.7601061. Epub 2006 Apr 6.
Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.
泛素（Ub）-蛋白连接酶（E3）常以多聚泛素（poly-Ub）链的形式用多个Ub分子修饰其底物。尽管结构不同的多聚Ub链（通过不同的Ub赖氨酸（Lys）残基连接）可赋予靶蛋白不同的命运，但关于E3如何选择用于链合成的Lys残基却知之甚少。在此，我们结合诱变、生物化学和质谱技术，来确定由KIAA10催化的链组装中连接选择的决定因素，KIAA10是一种HECT（与E6AP C末端同源）结构域E3，可合成K29和K48连接的链。聚焦于为链形成贡献Lys残基的Ub分子，我们发现与K48和K29相邻的特定表面残基对于链合成中各自Lys残基的使用至关重要。这种连接选择的直接机制与由Ubc9催化的SUMO化中底物位点选择机制相似，但不同于Mms2/Ubc13（Ub E2变体（UEV）/E2）复合物使用的链连接选择机制。
