Cherlyn Ng (left) Rebecca Jackson (Right) equal first authors.
Structural basis for a novel intrapeptidyl H-bond and reverse binding of c-Cbl-TKB domain substrates
Cherlyn Ng1, 3, Rebecca A Jackson2, 3, Jan P Buschdorf2, Qingxiang Sun1, Graeme R Guy2 and J Sivaraman1
1 Department of Biological Sciences, National University of Singapore, Singapore
2 Institute of Molecular and Cell Biology, Proteos, Singapore
3 Equal first authors
The c-Cbl tyrosine kinase binding domain (Cbl-TKB), essentially an 'embedded' SH2 domain, has a critical role in targeting proteins for ubiquitination. To address how this domain can bind to disparate recognition mofits and to determine whether this results in variations in substrate-binding affinity, we compared crystal structures of the Cbl-TKB domain complexed with phosphorylated peptides of Sprouty2, Sprouty4, epidermal growth factor receptor, Syk, and c-Met receptors and validated the binding with point-mutational analyses using full-length proteins. An obligatory, intrapeptidyl H-bond between the phosphotyrosine and the conserved asparagine or adjacent arginine is essential for binding and orientates the peptide into a positively charged pocket on c-Cbl. Surprisingly, c-Met bound to Cbl in the reverse direction, which is unprecedented for SH2 domain binding. The necessity of this intrapeptidyl H-bond was confirmed with isothermal titration calorimetry experiments that also showed Sprouty2 to have the highest binding affinity to c-Cbl; this may enable the selective sequestration of c-Cbl from other target proteins.
Published in The EMBO Journal advance online publication 14 February 2008;
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