Structural basis for ubiquitin-mediated dimerization and activation of the ubiquitin protein ligase Cbl-b.
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.
Department of Biochemistry, McGill University, Montréal, Canada.
Pascal
P
Peschard
Guennadi
G
Kozlov
Tong
T
Lin
I Ahmad
IA
Mirza
Albert M
AM
Berghuis
Stanley
S
Lipkowitz
Morag
M
Park
Kalle
K
Gehring
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D003460:Crystallization; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D050721:Proto-Oncogene Proteins c-cbl / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D025801:Ubiquitin; C492211:CBLB protein, human; D050721:Proto-Oncogene Proteins c-cbl