Tyrosine phosphorylation of the well packed ephrinB cytoplasmic beta-hairpin for reverse signaling. Structural consequences and binding properties.
Tyrosine phosphorylation of the 22-residue cytoplasmic region of ephrinB induces its binding to the SH2 domain of Grb4, thus initiating reverse signaling pathways controlling cytoskeleton assembly and remodeling. Recently, the region corresponding to this 22-residue motif was demonstrated to adopt a well packed beta-hairpin structure with a high conformational stability in the unphosphorylated cytoplasmic subdomain. However, because the binding to Grb4 is phosphorylation-dependent and the hairpin contains three conserved tyrosine residues that may be phosphorylated, the key events remain unknown as to how tyrosine phosphorylation affects the structure of this well packed beta-hairpin and which phosphorylation site is relevant to SH2 domain binding. By characterizing the structural and binding properties of six 22-residue SH2 domain-binding motifs with different phosphorylated sites, the present study reveals that, as shown by circular dichroism and NMR, the unphosphorylated 22-residue motif adopts a well formed beta-hairpin structure in isolation from the ephrinB cytoplasmic subdomain. However, this beta-hairpin is radically abolished by tyrosine phosphorylation, regardless of the relative location and number of Tyr residues. Unexpectedly, the peptides with either Tyr304 or Tyr316 phosphorylated show high affinity binding to SH2 domain, whereas the peptide with Tyr311 phosphorylated has no detectable binding. This implies that ephrinB with Tyr311 phosphorylated might have a currently unidentified binding partner distinct from the Grb4 protein, because Tyr311 is known to be phosphorylated in vivo. Based on the results above, it is thus proposed that the disruption of the tight side-chain packing by tyrosine phosphorylation in the well structured region of a signaling protein may represent a general activation mechanism by which a cryptic binding site is disclosed for new protein-protein interactions.
Song
Jianxing
J
Departments of Biochemistry and Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore. bchsj@nus.edu.sg
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D036342:Ephrins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D015513:Oncogene Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D036342:Ephrins; C485544:NCK2 protein, human; D015513:Oncogene Proteins; D014443:Tyrosine