Differential ubiquitin binding of the UBA domains from human c‐Cbl and Cbl‐b: NMR structural and biochemical insights
Differential ubiquitin binding of the UBA domains from human c‐Cbl and Cbl‐b: NMR structural and biochemical insights
复制标题
DOI：
10.1110/ps.036384.108
复制
发表时间：
2008-10
期刊：
影响因子：
8
通讯作者：
Zi-ren Zhou;Hong-Chang Gao;Chenjie Zhou;Yong-Gang Chang;Jing Hong;A. Song;D. Lin;Hong-Yu Hu
中科院分区：
文献类型：
--
作者：
Zi-ren Zhou;Hong-Chang Gao;Chenjie Zhou;Yong-Gang Chang;Jing Hong;A. Song;D. Lin;Hong-Yu Hu
Cbl 蛋白（RING 型 E3 泛素连接酶）负责泛素化激活的酪氨酸激酶并靶向它们进行降解。 c-Cbl和Cbl-b的C末端都有一个UBA（泛素相关）结构域，并且这两个UBA结构域具有很高的序列相似性（75%）。然而，只有来自 Cbl-b 的 UBA 可以结合泛素 (Ub)，而来自 c-Cbl 的 UBA 则不然。为了了解 UBA 结构域与具有不同亲和力的 Ub 特异性相互作用的机制，我们确定了这两个 UBA 结构域（来自人类 c-Cbl 的 cUBA 和来自 Cbl-b 的 UBAb）的溶液 NMR 结构。它们的结构表明这两个 UBA 结构域共享相同的折叠，即紧凑的三螺旋束，与典型的 UBA 折叠高度相似。化学位移扰动实验表明，UBAb 的螺旋-1 和环-1 形成了一个主要用于 Ub 结合的疏水表面。通过比较 UBAb 上的 Ub 相互作用表面和 cUBA 上的对应表面，我们发现 cUBA 上的疏水斑块被带负电的残基 Glu12 打断。荧光滴定数据显示UBAb的Ala12Glu突变体完全丧失了结合Ub的能力，而破坏二聚化的突变对Ub结合没有显着影响。这项研究提供了对 Cbl UBA 结构域的 Ub 结合特异性的结构和生化见解，其中第一螺旋上的疏水表面分布在它们对 Ub 结合的不同亲和力中起着至关重要的作用。也就是说，helix-1区域的氨基酸残基多样性，而不是二聚化，决定了各个UBA结构域与Ub结合的能力。
The Cbl proteins, RING‐type E3 ubiquitin ligases, are responsible for ubiquitinating the activated tyrosine kinases and targeting them for degradation. Both c‐Cbl and Cbl‐b have a UBA (ubiquitin‐associated) domain at their C‐terminal ends, and these two UBA domains share a high sequence similarity (75%). However, only the UBA from Cbl‐b, but not from c‐Cbl, can bind ubiquitin (Ub). To understand the mechanism by which the UBA domains specifically interact with Ub with different affinities, we determined the solution NMR structures of these two UBA domains, cUBA from human c‐Cbl and UBAb from Cbl‐b. Their structures show that these two UBA domains share the same fold, a compact three‐helix bundle, highly resembling the typical UBA fold. Chemical shift perturbation experiments reveal that the helix‐1 and loop‐1 of UBAb form a predominately hydrophobic surface for Ub binding. By comparing the Ub‐interacting surface on UBAb and its counterpart on cUBA, we find that the hydrophobic patch on cUBA is interrupted by a negatively charged residue Glu12. Fluorescence titration data show that the Ala12Glu mutant of UBAb completely loses the ability to bind Ub, whereas the mutation disrupting the dimerization has no significant effect on Ub binding. This study provides structural and biochemical insights into the Ub binding specificities of the Cbl UBA domains, in which the hydrophobic surface distribution on the first helix plays crucial roles in their differential affinities for Ub binding. That is, the amino acid residue diversity in the helix‐1 region, but not the dimerization, determines the abilities of various UBA domains binding with Ub.