title: 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 orients 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.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Cherlyn
authors.initials: C
authors.lastname: Ng
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Jackson
authors.forename: Jan P
authors.initials: JP
authors.lastname: Buschdorf
authors.forename: Qingxiang
authors.initials: Q
authors.lastname: Sun
authors.forename: Graeme R
authors.initials: GR
authors.lastname: Guy
authors.forename: J
authors.initials: J
authors.lastname: Sivaraman
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins; D010748:Phosphopeptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000737:chemistry* / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met; D017978:Receptors, Growth Factor / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; C485561:SPRY2 protein, human; D050721:Proto-Oncogene Proteins c-cbl; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met