title: Differential ubiquitin binding of the UBA domains from human c-Cbl and Cbl-b: NMR structural and biochemical insights.
abstract: 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.
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Zi-Ren
authors.initials: ZR
authors.lastname: Zhou
authors.forename: Hong-Chang
authors.initials: HC
authors.lastname: Gao
authors.forename: Chen-Jie
authors.initials: CJ
authors.lastname: Zhou
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Chang
authors.forename: Jing
authors.initials: J
authors.lastname: Hong
authors.forename: Ai-Xin
authors.initials: AX
authors.lastname: Song
authors.forename: Dong-Hai
authors.initials: DH
authors.lastname: Lin
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D050721:Proto-Oncogene Proteins c-cbl / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D025801:Ubiquitin; D050721:Proto-Oncogene Proteins c-cbl