Structural Basis for Ubiquitin-Mediated Dimerization and Activation of the Ubiquitin Protein Ligase Cbl-b

Abstract

Cbl proteins are E3 ubiquitin ligases that are negative regulators of many receptor tyrosine kinases. Cbl-b and c-Cbl contain a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Despite high sequence identity, Cbl UBA domains display remarkably different ubiquitin-binding properties. Here, we report the crystal structure of the UBA domain of Cbl-b in complex with ubiquitin at 1.9 A resolution. The structure reveals an atypical mechanism of ubiquitin recognition by the first helix of the UBA. Helices 2 and 3 of the UBA domain form a second binding surface, which mediates UBA dimerization in the crystal and in solution. Site-directed mutagenesis demonstrates that Cbl-b dimerization is regulated by ubiquitin binding and required for tyrosine phosphorylation of Cbl-b and ubiquitination of Cbl-b substrates. These studies demonstrate a role for ubiquitin in regulating biological activity by promoting protein dimerization.

Structural Basis for UBA-mediated Dimerization of c-Cbl Ubiquitin Ligase

“…This leads to significant differences in the buried surface for the Cbl-b and c-Cbl UBA dimers, which are 491 and 1050 Å 2 , respectively. In agreement with the lower buried surface, Cbl-b UBA dimerizes weakly in solution with K d of ϳ230 M (27), whereas c-Cbl shows a stronger dimerization with K d likely to be in low micromolar to high nanomolar range. In vitro, dimerization of the Cbl-b UBA domain is promoted by polyubiquitin binding (27), demonstrating that dimerization is a general property of Cbl UBA domains.…”

“…Not surprisingly, helix 3 is also responsible for dimerization of Cbl-b UBA (Fig. 6B) (27). In contrast to the c-Cbl UBA domain, in the Cbl-b UBA dimer, the helices 3 of both protomers stack almost parallel to each other, whereas second helices provide a much smaller contribution to the dimerization interface.…”

“…NMR samples contained 0.5-1.0 mM protein in 50 mM phosphate, 100 mM NaCl, pH 6.8. NMR sample preparation and signal assignments for the Cbl-b UBA domain are described elsewhere (27). For heterodimerization NMR titrations, unlabeled UBA domain from Cbl-b or c-Cbl was added to the 15 N-labeled 0.25-0.5 mM c-Cbl or Cbl-b UBA, respectively, to the final molar ratio of (6 Ϭ 7) to 1.…”

LRIG1 negatively regulates the oncogenic EGF receptor mutant EGFRvIII

“…As Cbl tyrosine phosphorylation is a prerequisite for its action as an ubiquitin ligase (Peschard et al, 2007), we examined whether Cbl was tyrosine phosphorylated and therefore competent to participate in LRIG1-mediated EGFRvIII degradation. Endogenous Cbl was immunoprecipitated and blotted with an antiphosphotyrosine antibody.…”

An E2 Accessory Domain Increases Affinity for the Anaphase-promoting Complex and Ensures E2 Competition

“…However, previous studies indicate that the UBA domain does not bind the ubiquitin covalently linked to the UBC domain (the donor ubiquitin) (26) or the ubiquitin attacking the E2-ubiquitin conjugate (the acceptor ubiquitin) (3). UBA domains in other proteins have been shown to bind non-ubiquitin folds (27)(28)(29). It therefore remains unclear how a putative ubiquitin-binding domain promotes a productive interaction between Ubc1 and the APC/C.…”