Tyrosine Phosphorylation of the Well Packed EphrinB Cytoplasmic β-Hairpin for Reverse Signaling

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 β-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 β-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 β-hairpin structure in isolation from the ephrinB cytoplasmic subdomain. However, this β-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. 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 β-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 β-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 β-hairpin structure in isolation from the ephrinB cytoplasmic subdomain. However, this β-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. The Eph 1The abbreviations used are: Eph, ephrin receptor; GST, glutathione S-transferase; HPLC, high-performance liquid chromatography; CD, circular dichroism; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single-quantum coherence spectroscopy; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. 1The abbreviations used are: Eph, ephrin receptor; GST, glutathione S-transferase; HPLC, high-performance liquid chromatography; CD, circular dichroism; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single-quantum coherence spectroscopy; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. receptors, with a total of 14 members, is the largest known family of receptor tyrosine kinases. They are implicated to function at the interface between pattern development and morphogenesis, such as axon guidance, cell migration, segmentation, and angiogenesis (1Holder N. Klein R. Development. 1999; 126: 2033-2044Google Scholar, 2Flanagan J.G. Vanderhaeghen P. Annu. Rev. Neurosci. 1998; 21: 309-345Google Scholar, 3Adams R.H. Klein R. Trends Cardiovasc. Med. 2000; 10: 183-188Google Scholar, 4Kullander K. Mather N.K. Diella F. Dottori M. Boyd A.W. Klein R. Neuron. 2001; 29: 73-84Google Scholar, 5Mellitzer G. Xu Q. Wilkinson D.G. Curr. Opin. Neurobiol. 2000; 10: 400-408Google Scholar). The signaling network mediated by Eph receptors and their ligand ephrin is conserved among metazoans, and eight mammalian ephrins have been identified so far. These can be grouped into two structural and functional families, ephrinA and ephrinB. EphrinB and their Eph receptors are all plasma membrane-anchored proteins and are unique in their ability to transmit signals bidirectionally, thus mediating contact-dependent cell-cell interactions for guidance and assembly of cells (4Kullander K. Mather N.K. Diella F. Dottori M. Boyd A.W. Klein R. Neuron. 2001; 29: 73-84Google Scholar, 5Mellitzer G. Xu Q. Wilkinson D.G. Curr. Opin. Neurobiol. 2000; 10: 400-408Google Scholar, 6Kullander K. Klein R. Nat. Rev. Mol. Cell. Biol. 2002; 3: 475-486Google Scholar, 7Cowan C.A. Henkemeyer M. Trends Cell Biol. 2002; 12: 339-346Google Scholar). Recently, the Eph-ephrinB-mediated signaling network has been found to be involved in learning and memory formation (8Murai K.K. Pasquale E.B. Neuron. 2002; 33: 159-162Google Scholar), and differential expressions of ephrinB were also correlated with tumorigenesis (9Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Google Scholar). The cytoplasmic tail of the ephrinB proteins plays a central role in mediating reverse signaling via protein-protein interactions with intracellular protein binding partners (10Cowan C.A. Henkemeyer M. Nature. 2001; 413: 174-179Google Scholar, 11Lu Q. Sun E.E. Klein R.S. Flanagan J.G. Cell. 2001; 105: 69-79Google Scholar). In particular, interaction between the 22-residue cytoplasmic region of ephrinB and the Grb4 SH2 domain initiates down-stream signaling pathways regulating cytoskeleton assembly and remodeling (7Cowan C.A. Henkemeyer M. Trends Cell Biol. 2002; 12: 339-346Google Scholar, 10Cowan C.A. Henkemeyer M. Nature. 2001; 413: 174-179Google Scholar). Recently, we determined the NMR structure of the ephrinB cytoplasmic subdomain, and showed that, in the absence of the tyrosine phosphorylation, this 22-residue motif adopted a well packed β-hairpin structure (Fig. 1) with a high conformational stability as indicated by 15N backbone dynamics data (12Song J. Vranken W. Xu P. Gingras R. Noyce R.S. Yu Z. Shen S.H. Ni F. Biochemistry. 2002; 41: 10942-10949Google Scholar). However, because the interaction between this 22-residue region and the Grb4 SH2 domain is phosphorylation-dependent (7Cowan C.A. Henkemeyer M. Trends Cell Biol. 2002; 12: 339-346Google Scholar, 10Cowan C.A. Henkemeyer M. Nature. 2001; 413: 174-179Google Scholar), and the β-hairpin region contains three conserved Tyr residues Tyr304, Tyr311, and Tyr316 (Fig. 1), the role of tyrosine phosphorylation in this interaction still remains unknown. On the other hand, although 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 (13Marks F. Protein Phosphorylation. VCH Verlagsgesellschaft, Wenheim, Germany1996Google Scholar). In this regard, the 22-residue ephrinB β-hairpin represents a unique model for addressing the structural consequence of tyrosine phosphorylation because: 1) it adopts a well packed β-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, thus offering the possibility of studying the consequence of multiple tyrosine phosphorylations. By characterizing structural and binding properties of a set of six 22-residue 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. The set of peptides includes one unphosphorylated, three single-, one double-, and one triple-phosphorylated forms. Detailed CD and NMR characterization of the six 22-residue peptides demonstrates that the isolated SH2-domain binding motif adopts a well packed β-hairpin structure in the unphosphorylated state as described before (12Song J. Vranken W. Xu P. Gingras R. Noyce R.S. Yu Z. Shen S.H. Ni F. Biochemistry. 2002; 41: 10942-10949Google Scholar), but this β-hairpin structure is dramatically abolished by tyrosine phosphorylations. Furthermore, NMR-based binding study indicates that the in vivo unphosphorylated site Tyr304 is actually 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 β-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. Cloning and Expression of the Grb4 SH2 Domain—A DNA fragment encoding human Grb4 SH2 domain (residues 283–380) was generated from Grb4 DNA by PCR with the forward primer, 5′-GGG GAA TTC AGA GAG TGG TAC TAC GGG-3′, and a reverse primer, 5′-GGG CTC GAG TCA CTG CAG GGC CCT GAC G-3′. The PCR product was cloned into the pGEX-5X-1 vector (Amersham Biosciences) using EcoRI/XhoI restriction sites, and the DNA sequence was verified by automated DNA sequencing. The expression construct for the SH2 domain was transformed into the Escherichia coli strain BL21 to express GST fusion protein and was subsequently purified using glutathione-Sepharose (Amersham Biosciences). The cleavage of the fusion protein was performed by incubating the fusion proteins with bovine factor Xa for 2 days. The purified SH2 domain protein was obtained by rebinding the released GST protein to the glutathione-Sepharose. For heteronuclear NMR experiments the SH2 domain protein were prepared in 15N-labeled form using a similar expression protocol except for growing E. coli cells in minimal M9 media instead of the 2YT media, with an addition of (15NH4)2SO4 for 15N labeling. Peptide Synthesis and Purification—Six 22-residue peptides with different sites and numbers of phosphorylations at Tyr304, Tyr311, and Tyr316 were synthesized using standard Fmoc (N-(9-fluorenyl)methoxy-carbonyl) chemistry. This set of peptides includes one unphosphorylated native peptide with a sequence of 301CPHYEKVSGDYGHPVYIVQEMP322 corresponding to the residues 301–322 of ephrinB2 (designated as ephrinB2301–322); three single-phosphorylated peptides, [pY304] ephrinB2301–322, [pY311]ephrinB2301–322, and [pY316]ephrinB2301–322, with phosphorylation at Tyr304, Tyr311, and Tyr316, respectively; one double-phosphorylated peptide [pY311,pY316]ephrinB2301–322; and one triple-phosphorylated peptide [pY304,pY311,pY316]ephrinB2301–322. For further mapping of the specificity and affinity determination of the ephrinB and SH2-domain interaction, three short phosphorylated peptides derived from the ephrinB2 sequence, pY304EKVSG, pY311GHPV, and pY316IVQEM, were also chemically synthesized using the same procedure. The peptides were purified by a reverse-phase high-performance liquid chromatography C18 column, and their identities were verified by MALDI-TOF mass spectrometry and NMR resonance assignments. Circular Dichroism Spectroscopy—CD experiments were performed on a Jasco J-715 spectropolarimeter, with samples containing 50 μm peptides in 20 at CD were at 20 using with a were for NMR and NMR of the 15N-labeled SH2 domain with a of was prepared by the protein into 50 of the 22-residue peptides with of were prepared by the peptides in of either or containing 50 or was to a of to the formation of The signal for the NMR was by the addition of 50 of of was also to the of by the procedure. NMR J. P. J. Scholar), D.G. J. Scholar), and M. J. NMR 1999; were on a of was used for NOESY, and was used for and were using the and for the peptides were through identification of in the with NOE in the G. K. J. Mol. Biol. Scholar, K. NMR of and and Scholar). The structural model of was generated from the corresponding region of the NMR structure (12Song J. Vranken W. Xu P. Gingras R. Noyce R.S. Yu Z. Shen S.H. Ni F. Biochemistry. 2002; 41: 10942-10949Google by using the of Tyrosine by Circular into the structure changes by tyrosine phosphorylation of the peptides, circular dichroism (CD) of all six 22-residue peptides were at and 20 in the CD of the unphosphorylated has two at and at The of a at and a one in the range of indicates a well formed or β-hairpin Circular Dichroism and the of Scholar). This that the 22-residue SH2-domain binding motif still adopts a well formed β-hairpin structure on its in isolation from the functional (12Song J. Vranken W. Xu P. Gingras R. Noyce R.S. Yu Z. Shen S.H. Ni F. Biochemistry. 2002; 41: 10942-10949Google Scholar). all three single-phosphorylated CD and with the to which are of or Circular Dichroism and the of Scholar). CD were also for the and triple-phosphorylated results that, upon tyrosine phosphorylation, the well formed β-hairpin adopted by 22-residue was regardless of the and numbers of the phosphorylation of Tyrosine by NMR further the detailed structural changes by tyrosine all six peptides were by NMR spectroscopy. The were for all six peptides by the of and in to well G. K. J. Mol. Biol. Scholar, K. NMR of and and Scholar), and the are in the the between the isolated 22-residue unphosphorylated peptide and the corresponding region in (12Song J. Vranken W. Xu P. Gingras R. Noyce R.S. Yu Z. Shen S.H. Ni F. Biochemistry. 2002; 41: 10942-10949Google Scholar). was for the to the resonance three residues Tyr311, and showed that the structure of the unphosphorylated is to that of the corresponding region in which is with the CD the of between and its phosphorylated forms. were among the sequence with changes changes were for (Fig. were three residues, and Tyr311 showing changes on a similar regardless of the of phosphorylated sites in the all phosphorylated peptides (Fig. it was shown that, and tyrosine phosphorylation no on the backbone conformation of model peptides J. J. 1999; Scholar). the current implies that tyrosine phosphorylations of ephrinB2301–322, regardless of their in the hairpin (Fig. 1), all similar structural which to the similar of in were for all six peptides to further detailed changes in side-chain packing upon tyrosine phosphorylation. show the same side-chain of of the six peptides in in the of a number of indicates that is well packed through it was isolated from the functional subdomain. further of NOE from with from the corresponding region in indicates that their are with CD and data shown in 2 and results of the NOE that the structure of the isolated is similar to that of the corresponding region in it is a well packed β-hairpin structure by extensive packing interactions among and the other (12Song J. Vranken W. Xu P. Gingras R. Noyce R.S. Yu Z. Shen S.H. Ni F. Biochemistry. 2002; 41: 10942-10949Google Scholar). In upon phosphorylation of Tyr304, extensive NOE (Fig. a disruption of the tight side-chain packing J. N. Nat. Biol. 1999; Scholar). phosphorylation of Tyr304 in a of to the of of Tyr304 also all NOE to the of as well as all between from the two hairpin such as between the and of Tyr316 and of or of (Fig. a and These indicated that a and disruption of the β-hairpin structure phosphorylation of Tyr311 structural as indicated by the of all to the of Tyr311, but also abolished all the long range side-chain packing, between from two hairpin similar to for (Fig. and These observations are similar to for [pY316]ephrinB2301–322, and triple-phosphorylated peptides (Fig. is to that, for the all to of Tyr304, Tyr311, and Tyr316 have dramatically (Fig. The of to the of the phosphorylated that the of a to the of the tyrosine residues may a or the or of which to a disruption of the side-chain packing the of the phosphorylated tyrosine residues. This is by the changes of CD upon tyrosine phosphorylation (Fig. 2) and NMR that, for all peptides, and of the phosphorylated can be in but to have it that the disruption of the tight side-chain packing to of three tyrosine is to the β-hairpin However, although tyrosine phosphorylation abolished all long range packing and range can still be identified the region (residues in all phosphorylated This is similar to in that this region might as an site in protein J. N. Nat. Biol. 1999; Scholar, E. M. M. G. Biochemistry. 2001; Scholar, J. Mol. Biol. Scholar, Annu. Rev. Scholar). of and binding properties of all six 22-residue ephrinB peptides for the Grb4 SH2 domain were by of the 15N-labeled SH2 domain with different The unphosphorylated native peptide no with a of the In the was also and showed no detectable binding activity with the results of biochemical (10Cowan C.A. Henkemeyer M. Nature. 2001; 413: 174-179Google Scholar). In of the 15N-labeled SH2 domain with the peptide showed that, the between the peptide and the SH2 domain was two of the were for many residues of the SH2 set is and set with extensive which have from the SH2 domain with the peptide the the set from the SH2 domain all to with the set from the SH2 addition of the peptide to a no further (Fig. This implies that the of the between the SH2 domain and [pY304] is the of For the is μm Biochemistry. 2002; 41: Scholar). However, as Biochemistry. 2002; 41: Scholar), and it would be important to the binding by other no detectable of the with a of the peptide that to the Grb4 SH2 For the peptide [pY316]ephrinB2301–322, at a two of the were for many residues of the SH2 set is and set with from the SH2 the was to the set from the SH2 domain to with the set from the SH2 domain, similar to that in Furthermore, the addition of the peptide to a no further (Fig. This indicates that the between the peptide and the SH2 domain is also the of the of the SH2 domain with is different from that of the SH2 domain with This may from different conformational changes by the binding of the two peptides to the same site on the SH2 domain, or from the binding of the two peptides to two different the that a of the of the 15N-labeled SH2 domain upon binding to either or [pY316]ephrinB2301–322, of the two can be by the mapping and would high of the domain to be determined by or NMR spectroscopy. Furthermore, the of the SH2 domain with the double-phosphorylated peptide was to that of the SH2 domain with the single-phosphorylated peptide (Fig. This that phosphorylation of Tyr311 binding. The of the SH2 domain with triple-phosphorylated peptide was to that of the SH2 domain with the single-phosphorylated peptide peptide (Fig. that the SH2 domain may to the triple-phosphorylated peptide in a similar as with the single-phosphorylated peptide the of the binding affinity and specificity between the phosphorylated ephrinB and the Grb4 SH2 domain, the binding interactions between the SH2 domain and three short phosphorylated peptides pY304EKVSG, pY311GHPV, and were by The peptide to the of a of of the 15N-labeled SH2 domain at a of and the it was at a of This that the between the SH2 domain and is with a binding affinity Biochemistry. 2002; 41: as with the interaction with the of the 15N-labeled SH2 domain in the of a of the peptide is similar to that of the 15N-labeled SH2 domain with that the short and peptides to the SH2 domain in a similar the of the results the of the high of the between the SH2 domain and the different phosphorylated For and pY316IVQEM, no detectable was with a of the peptides These results that, for the phosphorylation sites Tyr304 and Tyr316, residues present in the peptides and are to high affinity with the SH2 The ephrinB as signaling controlling such as axon guidance and functional have the role of the cytoplasmic domain in K. Klein R. Nat. Rev. Mol. Cell. Biol. 2002; 3: 475-486Google Scholar, 7Cowan C.A. Henkemeyer M. Trends Cell Biol. 2002; 12: 339-346Google Scholar, R.H. Diella F. F. Klein R. Cell. 2001; Scholar, Curr. Opin. Cell Biol. 2002; Scholar, Trends Neurosci. Scholar). of two intracellular binding partners of and Grb4 into the interactions the Eph-ephrinB-mediated reverse signaling network (7Cowan C.A. Henkemeyer M. Trends Cell Biol. 2002; 12: 339-346Google Scholar, 10Cowan C.A. Henkemeyer M. Nature. 2001; 413: 174-179Google Scholar, 11Lu Q. Sun E.E. Klein R.S. Flanagan J.G. Cell. 2001; 105: 69-79Google Scholar). it was proposed that by of the ephrinB cytoplasmic domain, the reverse signaling be and between two one to receptor signaling and phosphorylation-dependent that modulates dynamics M. R. Klein R. Mol. Cell. 2002; Scholar). the central role of tyrosine phosphorylation in Eph-ephrinB-mediated reverse it unknown how tyrosine phosphorylation affects the structure of the 22-residue SH2 domain binding motif with a well packed β-hairpin and which phosphorylation site is relevant to the SH2 domain binding. phosphorylation of proteins is the mechanism in controlling the biological activity of proteins by which for a of from of to of and expression (13Marks F. Protein Phosphorylation. VCH Verlagsgesellschaft, Wenheim, Germany1996Google Scholar, J. J. 1999; Scholar, Yu Pasquale E.B. J. Biol. 2001; Scholar, Annu. Rev. Scholar, Biochemistry. Scholar, J. Nat. Biol. Scholar, W. J. 2001; Scholar). has been that phosphorylation protein activity by conformational but the detailed remain poorly the structural consequence and binding properties of tyrosine phosphorylations on the well packed ephrinB cytoplasmic β-hairpin might contribute to our general knowledge how tyrosine phosphorylation signaling through conformational In the first of this the detailed CD and NMR characterization of a set of six 22-residue ephrinB peptides demonstrates that the unphosphorylated adopts a well packed β-hairpin structure as described (12Song J. Vranken W. Xu P. Gingras R. Noyce R.S. Yu Z. Shen S.H. Ni F. Biochemistry. 2002; 41: 10942-10949Google Scholar), but this structure is radically upon tyrosine phosphorylation, regardless of the of the phosphorylated many tyrosine in proteins in the formation of the packing by with other long range the of a with a side-chain and the is that of protein through disrupting the side-chain packing may represent a general mechanism by which tyrosine phosphorylation the of In the the phosphorylation of the 22-residue motif is further correlated to the binding interaction with the Grb4 SH2 The NMR study reveals that the in vivo unphosphorylated site Tyr304 and the in vivo phosphorylated site Tyr316 can the Grb4 SH2 domain with high Unexpectedly, the peptide with Tyr311 phosphorylated showed no detectable binding ability to the SH2 the phosphorylated Tyr304 can as an in vivo site for the Grb4 SH2 domain to be further the that the peptide with Tyr311 phosphorylation no binding activity to the Grb4 SH2 domain that phosphorylated Tyr311 might have other currently unknown in vivo binding because Tyr311 or Tyr316 are conserved tyrosine residues the 22-residue β-hairpin and was shown to be phosphorylated in vivo (7Cowan C.A. Henkemeyer M. Trends Cell Biol. 2002; 12: 339-346Google Scholar, M. R. Klein R. Mol. Cell. 2002; Scholar, Yu Pasquale E.B. J. Biol. 2001; Scholar). indicated that the protein formed a with either the unphosphorylated or phosphorylated ephrinB cytoplasmic domain M. J. Scholar), by a further of other this it would be of to the phosphorylation of Tyr311 to the protein-protein interactions. Based on current it that for the interaction between the ephrinB cytoplasmic region and the Grb4 SH2 domain, three have to be 1) the well packed hairpin structure has to be by tyrosine phosphorylation to the region for the Grb4 SH2 2) the is to provide the binding P. Trends Cell Biol. 2001; Scholar, J. Ni F. Cell Biol. 1998; and the the phosphorylated tyrosine residues have to be present to high affinity binding. The is by the results of NMR binding study on the interactions between the SH2 domain and three short peptides, which indicates that the high affinity binding between the SH2 domain and phosphorylated the short to the phosphorylated is also to that phosphorylation of Tyr311, which and but in no detectable binding to the Grb4 SH2 The that the peptide is to the Grb4 SH2 domain may provide a for the of disrupting the protein-protein interaction between ephrinB and the Grb4 protein Curr. Opin. Cell Biol. 2002; Scholar). In the present NMR study the first detailed picture showing how the well packed β-hairpin structure of ephrinB cytoplasmic domain can be dramatically by and phosphorylations at three conserved tyrosine residues. The NMR binding study further two of three tyrosine residues the 22-residue hairpin of ephrinB as phosphorylation sites relevant for high affinity binding to the Grb4 SH2 In particular, the that the 22-residue peptide with Tyr311 phosphorylated no binding to the Grb4 SH2 domain implies that ephrinB phosphorylated at Tyr311 might have currently unidentified in vivo binding partners other The current results can be to 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 the cryptic binding site is disclosed for further protein-protein interactions. the for which to the for of the Z. Yu for the with the of the Grb4 SH2 domain, J. for peptide and K. and at the Protein and of for to CD and for MALDI-TOF mass with

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