Abstract
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.
Highlights
Tyrosine phosphorylation of the 22-residue cytoplasmic region of ephrinB induces its binding to the SH2 domain of Grb4, initiating reverse signaling pathways controlling cytoskeleton assembly and remodeling
Tyrosine Phosphorylation of the EphrinB Cytoplasmic -Hairpin ephrinB peptides, the current study focuses on addressing the structural consequences of tyrosine phosphorylation and on identification of phosphorylation sites relevant to the SH2 domain binding by use of circular dichroism (CD) and NMR spectroscopy
Structural Effects of Tyrosine Phosphorylations Characterized by Circular Dichroism—To gain insight into the globular secondary structure changes caused by tyrosine phosphorylation of the peptides, far-UV circular dichroism (CD) spectra of all six 22-residue peptides were collected at pH 6.8 and 20 °C
Summary
Ephrin receptor; GST, glutathione S-transferase; HPLC, high-performance liquid chromatography; CD, to function at the interface between pattern development and morphogenesis, such as axon guidance, cell migration, segmentation, and angiogenesis [1,2,3,4,5]. Selective and reversible phosphorylation plays a pivotal role in controlling biological activity of proteins in many biochemical processes, the mechanism still remains poorly understood especially in structural terms [13] In this regard, the 22-residue ephrinB -hairpin represents a unique model for addressing the structural consequence of tyrosine phosphorylation because: 1) it adopts a well packed -hairpin structure with extensive long range side-chain packing, which would facilitate the assessment of structural changes upon tyrosine phosphorylation and 2) it contains three conserved tyrosine residues, offering the possibility of studying the consequence of multiple tyrosine phosphorylations. NMR-based binding study indicates that the in vivo unphosphorylated site Tyr304 is relevant to the SH2 domain binding, whereas the in vivo phosphorylated site Tyr311 is irrelevant These results provide the first detailed picture showing how cryptic binding sites buried in a well packed -hairpin of a signaling protein is activated by disrupting the tight side-chain packing through tyrosine phosphorylation. These observations may bear important implications in deciphering the Eph-ephrinB-mediated reverse signaling mechanism and contribute to our general knowledge of how tyrosine phosphorylation modulates signal transduction by switching the conformation of a signaling protein
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