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 (ubiquitinassociated) 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.

Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling

“…Although few other UBA dimers have been identified or characterised, dimerisation of the UBA domains of the scaffold proteins c-Cbl and Cbl-b, [25][26][27] coupled with their capacity to control signalling downstream from many receptor-tyrosine kinases, suggests that dimerisation provides a mechanism for an expanded repertoire of Cbl-dependent signals. NMR titration studies estimated c-Cbl homodimerisation to be in the low micromolar affinity range; however, binding studies with an excess of Ub failed to produce any detectable interaction in HSQC experiments, suggesting that the functional role of the c-Cbl is not in regulating Ub interactions.…”

“…24 More recently, dimers have been characterised for the UBA domains of c-Cbl and Cbl-b involving hydrophobic surface interactions at the interface of helices 2 and 3. [25][26][27] The role of c-Cbl and Cbl-b as scaffold proteins, coupled with their capacity to interact with different downstream signalling proteins, suggests that dimerisation provides a mechanism for an expanded repertoire of Cbl-dependent signals recruited to receptor-tyrosine kinases. Although, in the case of the c-Cbl homodimer, Ub recognition does not appear to be its primary function, 25 the dimerisation and activation of the Ub protein ligase Cbl-b are Ub-mediated.…”

Inhibition of SRC Corrects GM-CSF Hypersensitivity That Underlies Juvenile Myelomonocytic Leukemia

“…Cbl is a RING-type E3 ubiquitin ligase that ubiquitylates receptor and non-receptor tyrosine kinases (8). Cbl contains a highly conserved N-terminal tyrosine kinase-binding (TKB) domain that mediates interaction between Cbl and phosphorylated tyrosines on activated receptor tyrosine kinases such as epidermal growth factor receptor (EGFR; ref.…”

A Helical Conotoxin from Conus imperialis Has a Novel Cysteine Framework and Defines a New Superfamily

“…A search using DALI (20), which interrogates the Protein Data Bank data base for structural homologues, showed that im23a had structural similarities to the ubiquitin-associated domain of the ubiquitin ligase Cbl-b (PDB ID 2DO6), which exists naturally as a dimer (21,22). An overlay of the two structures shows that the first two helices across the hairpin motif matched well with each other.…”

“…6C). The ubiquitinassociated domain consists of a compact three-helix bundle stabilized by a hydrophobic core (22); unlike im23a, the ubiquitin-associated domain does not contain any disulfide bonds to stabilize the structure. Several aspects of the NMR data for im23a (amide exchange rates, 3 J HNHA values, backbone chemical shifts, lack of medium-range NOEs) show quite clearly that its C-terminal region is not helical under the solution conditions examined here, and the Cys-26 to Cys-41 disulfide bridge dictates that the orientation of the C-terminal region is distinct from that in Cbl-b.…”