Lin H, Wing S S
Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada.
J Biol Chem. 1999 May 21;274(21):14685-91. doi: 10.1074/jbc.274.21.14685.
The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1. E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain.
泛素（Ub）缀合酶（E2s）决定底物特异性的结构基础仍不清楚。我们克隆了兔网织红细胞E217K，因为与大小相似的I类E2s（E214K和UBC4）不同，它无法支持泛素 - 蛋白连接酶（E3）依赖的与内源性蛋白的缀合。RNA分析显示，这种E2在所有测试组织中均有表达，在睾丸中的表达水平更高。对不同年龄大鼠睾丸RNA的分析表明，E217K mRNA在第15天到第30天被诱导表达。预测的氨基酸序列表明E217K是一种19.5 kDa的I类E2，但与其他I类酶不同，它在活性位点半胱氨酸远端有一个13个氨基酸的插入。E217K与酿酒酵母UBC7的氨基酸同一性为74%，因此，我们将其重新命名为哺乳动物UBC7。酵母UBC7晶体结构表明，该插入在原本保守的折叠结构之外形成了一个环。此前对E2s的序列分析表明，这个环是一个高变区，可能在底物特异性中起作用。我们创建了缺失该环的突变体UBC7（ubc7Deltaloop）和带有插入环的突变体E214k（E214k+loop），并对它们的生化功能进行了表征。ubc7Deltaloop对E1-Ub硫酯的亲和力高于天然UBC7，并允许Ub与睾丸中的选定蛋白缀合，但不允许与内源性网织红细胞蛋白进行广泛的E3依赖缀合。令人惊讶的是，E214k+loop无法从泛素激活酶（E1）接受Ub，但能够从E1接受NEDD8。E
