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

EMBO J. 2008 Mar 5;27(5):804-16. doi: 10.1038/emboj.2008.18. Epub 2008 Feb 14.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Line
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Membrane Proteins
  • Phosphopeptides / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-cbl / chemistry*
  • Proto-Oncogene Proteins c-cbl / metabolism*
  • Proto-Oncogene Proteins c-met
  • Receptors, Growth Factor / metabolism
  • src Homology Domains

Substances

  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Phosphopeptides
  • Proto-Oncogene Proteins
  • Receptors, Growth Factor
  • SPRY2 protein, human
  • Proto-Oncogene Proteins c-cbl
  • MET protein, human
  • Proto-Oncogene Proteins c-met