Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
Biochemistry. 2012 May 22;51(20):4175-87. doi: 10.1021/bi300058m. Epub 2012 May 14.
Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2Ub conjugates have been shown to form a range of structures in solution. In addition, select E2Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5cUb conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5cUb oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.
蛋白质的泛素化（Ub）后修饰调节了许多细胞过程，包括蛋白质质量控制、DNA 修复、内吞作用和细胞信号转导。在泛素化级联反应中，Ub 的 C 末端与泛素结合酶（E2）的活性位点半胱氨酸之间形成硫酯键连接的共轭物。E2Ub 共轭物与泛素连接酶（E3）相互作用，将 Ub 转移到靶蛋白上的赖氨酸残基上。柔性连接的 E2Ub 共轭物已被证明在溶液中形成一系列结构。此外，选择的 E2Ub 共轭物通过 Ub 和不同共轭物的 E2 成分之间的非共价“背面”相互作用而聚合。需要进一步的研究来弥合动态单体共轭物、E2Ub 寡聚物以及泛素化机制之间的差距。我们提出了一个新的 2.35Å 分辨率的寡聚 UbcH5cUb 共轭物晶体结构。该共轭物形成交错的线性寡聚物，与之前报道的唯一寡聚共轭物的“无限螺旋”螺旋排列有很大的不同。我们的结构在共轭物构象上也与其他结构特征化的共轭物不同。尽管存在这些差异，但我们发现背面相互作用模式在不同的共轭物寡聚体中是保守的，并且不依赖于共轭物内相对 E2-Ub 取向。我们描绘了 Ub 上的一个共同的共轭物内 E2 结合表面。此外，我们证明了 E3 CHIP（Hsp70 相互作用蛋白羧基末端）直接与 UbcH5cUb 寡聚物相互作用，而不仅与共轭物单体相互作用。这些结果提供了对 E2~Ub 共轭物和共轭物寡聚物构象多样性的深入了解，以及它们与 E3 的兼容性和相互作用，这对泛素化过程有重要影响。
