Structural basis for a novel intrapeptidyl H-bond and reverse binding of c-Cbl-TKB domain substrates

Abstract

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.