Zhou Zi-Ren, Gao Hong-Chang, Zhou Chen-Jie, Chang Yong-Gang, Hong Jing, Song Ai-Xin, Lin Dong-Hai, Hu Hong-Yu
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Protein Sci. 2008 Oct;17(10):1805-14. doi: 10.1110/ps.036384.108. Epub 2008 Jul 2.
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.
Cbl蛋白属于RING型E3泛素连接酶，负责将活化的酪氨酸激酶泛素化并使其靶向降解。c-Cbl和Cbl-b在其C末端均具有一个UBA（泛素相关）结构域，这两个UBA结构域具有高度的序列相似性（75%）。然而，只有Cbl-b的UBA结构域能够结合泛素（Ub），而c-Cbl的UBA结构域则不能。为了理解UBA结构域以不同亲和力与Ub特异性相互作用的机制，我们测定了这两个UBA结构域的溶液核磁共振结构，即来自人c-Cbl的cUBA和来自Cbl-b的UBAb。它们的结构表明，这两个UBA结构域具有相同的折叠方式，即紧密的三螺旋束，与典型的UBA折叠非常相似。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上与Ub相互作用的表面及其在cUBA上的对应表面，我们发现cUBA上的疏水补丁被带负电荷的残基Glu12打断。荧光滴定数据表明，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结合没有显著影响。这项研究为Cbl UBA结构域的Ub结合特异性提供了结构和生化方面的见解，其中第一个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关键作用。也就是说，螺旋-1区域的氨基酸残基多样性而非二聚化决定了各种UBA结构域与Ub结合的能力。
