Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Structure. 2010 Aug 11;18(8):955-65. doi: 10.1016/j.str.2010.04.017.
Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.
人源 E4B，也称为 UFD2a，是一种含有 U 盒的蛋白质，作为 E3 泛素连接酶和 E4 多泛素链延伸因子发挥作用。E4B 被认为通过与伴侣蛋白结合参与错误折叠或受损蛋白质的蛋白酶体降解。U 盒结构域是 E2 泛素连接酶的锚定位点，但对其结合机制知之甚少。本研究通过 X 射线晶体学和 NMR 光谱学，确定了 E4B U 盒游离态和与 UbcH5c 和 Ubc4 E2 结合态的结构。先前表征的 U 盒结构域为同源二聚体，而我们发现 E4B U 盒是通过氢键网络稳定的单体，这些氢键是通过标量耦合测量确定的。这些结构研究，辅之以基于量热法和 NMR 的结合测定，提示 E4B U 盒对 UbcH5c 和 Ubc4 进行变构调节，并为理解涉及 E4B 的泛素化机制如何组装提供了分子基础。
