Surface Loops in a Single SH2 Domain Are Capable of Encoding the Spectrum of Specificity of the SH2 Family*

Src homology 2 (SH2) domains play an essential role in cellular signal transduction by binding to proteins phosphorylated on Tyr residue. Although Tyr phosphorylation (pY) is a prerequisite for binding for essentially all SH2 domains characterized to date, different SH2 domains prefer specific sequence motifs C-terminal to the pY residue. Because all SH2 domains adopt the same structural fold, it is not well understood how different SH2 domains have acquired the ability to recognize distinct sequence motifs. We have shown previously that the EF and BG loops that connect the secondary structure elements on an SH2 domain dictate its specificity. In this study, we investigated if these surface loops could be engineered to encode diverse specificities. By characterizing a group of SH2 variants selected by different pY peptides from phage-displayed libraries, we show that the EF and BG loops of the Fyn SH2 domain can encode a wide spectrum of specificities, including all three major specificity classes (p + 2, p + 3 and p + 4) of the SH2 domain family. Furthermore, we found that the specificity of a given variant correlates with the sequence feature of the bait peptide used for its isolation, suggesting that an SH2 domain may acquire specificity by co-evolving with its ligand. Intriguingly, we found that the SH2 variants can employ a variety of different mechanisms to confer the same specificity, suggesting the EF and BG loops are highly flexible and adaptable. Our work provides a plausible mechanism for the SH2 domain to acquire the wide spectrum of specificity observed in nature through loop variation with minimal disturbance to the SH2 fold. It is likely that similar mechanisms may have been employed by other modular interaction domains to generate diversity in specificity. Src homology 2 (SH2) domains play an essential role in cellular signal transduction by binding to proteins phosphorylated on Tyr residue. Although Tyr phosphorylation (pY) is a prerequisite for binding for essentially all SH2 domains characterized to date, different SH2 domains prefer specific sequence motifs C-terminal to the pY residue. Because all SH2 domains adopt the same structural fold, it is not well understood how different SH2 domains have acquired the ability to recognize distinct sequence motifs. We have shown previously that the EF and BG loops that connect the secondary structure elements on an SH2 domain dictate its specificity. In this study, we investigated if these surface loops could be engineered to encode diverse specificities. By characterizing a group of SH2 variants selected by different pY peptides from phage-displayed libraries, we show that the EF and BG loops of the Fyn SH2 domain can encode a wide spectrum of specificities, including all three major specificity classes (p + 2, p + 3 and p + 4) of the SH2 domain family. Furthermore, we found that the specificity of a given variant correlates with the sequence feature of the bait peptide used for its isolation, suggesting that an SH2 domain may acquire specificity by co-evolving with its ligand. Intriguingly, we found that the SH2 variants can employ a variety of different mechanisms to confer the same specificity, suggesting the EF and BG loops are highly flexible and adaptable. Our work provides a plausible mechanism for the SH2 domain to acquire the wide spectrum of specificity observed in nature through loop variation with minimal disturbance to the SH2 fold. It is likely that similar mechanisms may have been employed by other modular interaction domains to generate diversity in specificity. The Src homology 2 (SH2) 1The abbreviations used are:SH2Src homology 2CDRcomplementarity-determining loopsOPALoriented peptide array library. 1The abbreviations used are:SH2Src homology 2CDRcomplementarity-determining loopsOPALoriented peptide array library. domain, originally identified in the viral oncogene product v-fps/fes, was subsequently found in numerous metazoan proteins (1Pawson T. Scott J.D. Protein phosphorylation in signaling–50 years and counting.Trends Biochem. Sci. 2005; 30: 286-290Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 2Sadowski I. Stone J.C. Pawson T. A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.Mol. Cell. Biol. 1986; 6: 4396-4408Crossref PubMed Scopus (386) Google Scholar). It is known now that the human genome encodes ∼120 SH2 domains that are dispersed in more than 110 proteins. These include protein or lipid kinases, protein phosphatases, small GTPases, cytoskeleton regulators, and adaptor/scaffolding proteins and other regulators of signal transduction (3Huang H. Li L. Wu C. Schibli D. Colwill K. Ma S. Li C. Roy P. Ho K. Songyang Z. Pawson T. Gao Y. Li S.S. Defining the specificity space of the human SRC homology 2 domain.Mol. Cell. Proteomics. 2008; 7: 768-784Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 4Liu B.A. Jablonowski K. Raina M. Arcé M. Pawson T. Nash P.D. The human and mouse complement of SH2 domain proteins establishing the boundaries of phosphotyrosine signaling.Mol. Cell. 2006; 22: 851-868Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). SH2 domains exert their functions by binding to the phosphotyrosine (pY) residue embedded in specific sequence motifs, thereby enabling transduction of signals emanated from tyrosine kinases to downstream molecules (1Pawson T. Scott J.D. Protein phosphorylation in signaling–50 years and counting.Trends Biochem. Sci. 2005; 30: 286-290Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 5Pawson T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems.Cell. 2004; 116: 191-203Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 6Scott J.D. Pawson T. Cell communication: the inside story.Sci Am. 2000; 282: 72-79Crossref PubMed Scopus (65) Google Scholar). The importance of the tyrosine kinase-pY-SH2 signaling axis in normal physiology and disease pathogenesis is underscored by the fact that drugs targeting components of this axis form the largest collection of targeted therapeutics used in the clinic to treat cancer and other complex human diseases (7Wu P. Nielsen T.E. Clausen M.H. FDA-approved small-molecule kinase inhibitors.Trends Pharmacol. Sci. 2015; 36: 422-439Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar). Src homology 2 complementarity-determining loops oriented peptide array library. Src homology 2 complementarity-determining loops oriented peptide array library. SH2 domains, related to one another by structure and function, are ∼100-residue in length and fold into a globular structure comprising a central β-sheet (with strands βA to βG) flanked by two α-helices (αA and αB) (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google Scholar, 9Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.Nature. 1992; 358: 646-653Crossref PubMed Scopus (576) Google Scholar, 10Waksman G. Shoelson S.E. Pant N. Cowburn D. Kuriyan J. Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.Cell. 1993; 72: 779-790Abstract Full Text PDF PubMed Scopus (655) Google Scholar). A typical SH2 domain recognizes the pY and a specific residue C-terminal to the pY in a two-pronged plug two-holed socket mode (11Hwang P.M. Li C. Morra M. Lillywhite J. C. Pawson T. J.D. Li S.C. A binding mechanism for the SH2 domain: structural and to the J. PubMed Scopus Google Scholar, G. the plug two-holed for the mechanism of binding of the Src SH2 domain to phosphotyrosyl a PubMed Scopus Google Scholar). Although all SH2 domains a and the same mode of pY recognition (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google in specificity and mode of recognition for the C-terminal residue (3Huang H. Li L. Wu C. Schibli D. Colwill K. Ma S. Li C. Roy P. Ho K. Songyang Z. Pawson T. Gao Y. Li S.S. Defining the specificity space of the human SRC homology 2 domain.Mol. Cell. Proteomics. 2008; 7: 768-784Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar). on from a we the SH2 domains into three specificity p + 2, p + 3 and p + T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar). The p + 3 by the Src SH2 domain, a residue the p + 3 residue C-terminal to the pY The and SH2 domains, to the p + 2 and p + classes prefer peptides with an the p + 2 or a residue the p + T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar, N. Huang J. M. L. L. Pawson T. S. a protein interaction in cell Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, B.A. Nash P.D. The of SH2 domain interactions signal 2012; PubMed Scopus Google Scholar). The C-terminal specificity is by a binding or to as specificity the surface of an SH2 domain that the p + 2, p + 3 or p + residue in the peptide T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar). We have shown previously that two surface loops on the SH2 domain, the EF loop the and and the BG loop the and not in the of the specificity of the peptide to the N. Huang J. M. L. L. Pawson T. S. a protein interaction in cell Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In a typical SH2 domain, one of the three specificity is for binding the are of or by specific from the EF and BG in the Src SH2 domain (p + 3 the p + is by a residue from the BG loop in the SH2 domain (p + the p + 3 is by an EF loop residue. In the of the SH2 domain (p + 2 the p + 3 and p + binding are T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar). The role of the EF and BG loops in SH2 domain specificity was underscored in the that specificity of an SH2 domain may be or by these the residue in the Src SH2 domain from to in of specificity from the p + 3 to p + 2 Songyang Z. M.D. Pawson T. SH2 domain specificity and activity by a PubMed Scopus Google Scholar). In the residue in the p + SH2 domain with an a of specificity to the p + 3 T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar). Although the a role for the EF and BG loops in an can these surface loops encode the wide spectrum of found for the SH2 domain in a to the role of the complementarity-determining in the specificity of an J. L. J. and of loops in high Biol. Full Text Full Text PDF PubMed Scopus Google this we phage-displayed of the Fyn SH2 domain in the EF and BG loops in length and residue By the with with diverse we identified variants that a wide of specificities. peptide including and peptide and binding we the specificity and affinity for a of variants from the phage-displayed Our that the EF and BG loops are highly and of a wide spectrum of specificity found in SH2 The loop was by (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google Scholar, P. to the for and secondary 2012; PubMed Scopus Google Scholar). The was peptides on a with of to to was by the into a with and to to the of the SH2 variants (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google Scholar). on the on an peptide the with for and or for binding by (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google Scholar). A was the or and the peptide to the of and of the peptides by peptides in with in a for The was in to in for with and used The an a with a SH2 the peptide array was three in in and protein was to the and with the for The was three in and with a the was in and with a for in the The was in in the and with a and the embedded of the and the embedded of the The binding signal for a variant was as the of the for the binding signals the The of a variant domain for pY peptide is to the is the signal of is the of more than one residue in the was the of for was SH2 protein was in a by the of peptide in The in the for to on an with the and Binding by the binding to a the We employed the Fyn SH2 domain to if the EF and BG loops can encode a wide of specificity. The EF loop of the Fyn SH2 domain three its BG loop We two of the Fyn SH2 domain in the EF and BG loop by (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google Scholar, P. to the for and secondary 2012; PubMed Scopus Google The on the and for binding to pY peptides (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google the three major classes p + p + and p + The to the of variants by bait peptides on sequence diversity of the bait peptides and the we selected Fyn SH2 variants for of the variants identified by different bait in the bait peptide and loop are to their The variants selected for and are with the variant identified by bait peptides the identified by bait peptides that a residue the or A feature of the variants selected by the p + group of bait peptides is the for the EF of the identified an residue or the A residue likely the binding in a the residue in the SH2 domain (p + 2 T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar). encode p + specificity, it is to have the p + binding to be in by an with a or the or in the or Intriguingly, variants by the peptide the small the p + in to an p + a BG loop in the or In the same by the peptide (p + and variants with the BG loop or a BG loop the p + binding these are to have acquired p + specificity shown not a for the p + In variants selected by the p + peptide a or an residue the or and a residue the suggesting that these variants have the p + 3 specificity of the Fyn SH2 and of a of Fyn SH2 variants for different specificity p + p + p + p + p + in are from binding or binding to the peptides with an the sequence in a in are from binding or binding to the peptides with an the sequence the of specificities, we selected Fyn SH2 variants with distinct loop and in as The protein was used to an the sequence a of M. Li S.S. Songyang Z. oriented peptide array to Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We have previously employed the to the specificity of human SH2 domains (3Huang H. Li L. Wu C. Schibli D. Colwill K. Ma S. Li C. Roy P. Ho K. Songyang Z. Pawson T. Gao Y. Li S.S. Defining the specificity space of the human SRC homology 2 domain.Mol. Cell. Proteomics. 2008; 7: 768-784Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). A the and (3Huang H. Li L. Wu C. Schibli D. Colwill K. Ma S. Li C. Roy P. Ho K. Songyang Z. Pawson T. Gao Y. Li S.S. Defining the specificity space of the human SRC homology 2 domain.Mol. Cell. Proteomics. 2008; 7: 768-784Abstract Full Text Full Text PDF PubMed Scopus (178) Google is that the with and of on was in to A was used as the and a with as the secondary to the protein by The and in the as and shown in and variant a binding on the variant a for an the p + 2 suggesting that it to the p + The binding signals on an subsequently and the of was the signal the to a of for a given residue a specific and for of for the different variants on the on the shown in the variants and selected by the p + group of an residue the p + 2 for a small of variants that not show p + specificity, likely of in the BG loop by the a p + The of the the p + 3 and plug the p + an for the p + The that the of the bait peptide and the variants are related we as the for a variant to for a specificity on this of the variants selected by peptides the could be to the p + In of the variants by bait peptides this could be to the p + by the of variants with the p + 3 specificity from to the bait peptides the on the for p + and p + we the variants into to and specificity for p + than p + These variants all selected by peptides the In of and and selected by the the specificity to the Intriguingly, the variants and by bait peptides with and the Fyn and SH2 domains specificity for p + and p + on the a of binding to peptides with the or these that the loops in the Fyn SH2 domain can a wide spectrum of that of the bait complement the we the specificity of the loop variants by peptide The same used in the SH2 and a The peptide array was for binding to different variants and The binding signals and to generate the in the same as for the We found on the the the the affinity for a peptide with the the SH2 variants distinct with the bait peptides the or in the from the the of the and of the variants including and with the peptides In the variants and of the variants including and a for the peptides than the peptides Intriguingly, the Fyn and SH2 domains to of suggesting that these SH2 domains have specificities. The to the variants for to the or In this to the EF and BG loops that likely play an role in specificity T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar). the p + 2 of SH2 domain, it been shown that the peptide adopt a to with the residue N. Huang J. M. L. L. Pawson T. S. a protein interaction in cell Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). we found that the variants with the for the a residue Tyr or the Furthermore, the same variants an residue or the likely functions to plug the p + binding Intriguingly, the variants that the group with a p + (with one or more or or or the EF the Fyn and SH2 domains to this It is that the or in these variants the of with the of the p + 2 residue or the in the the EF loop may encode p + specificity a variety of different In to the of numerous variants with p + variants a for the than the Fyn SH2 domain that the Fyn SH2 domain is for p + 3 We that the variants for the p + specificity all an residue the suggesting that these variants the of these variants and with the p + Although the Fyn SH2 domain is not known to p + specificity, it a for bait peptides the Intriguingly, including and a for this than the Fyn SH2 shown the and variants to peptides with than the Fyn SH2 these that SH2 variants with distinct can be through in the EF and BG Although the and peptide to variants with that are different from that of the domain, it is to the binding in this we the of SH2 variants for peptides the or motifs by with proteins and We the Fyn SH2 domain for and the Src and SH2 domains as of the p + 2, p + and p + specificity The in affinity for a variant to the Fyn SH2 domain was used as a of specificity the same peptide ligand. In with the peptide array we found that the Fyn SH2 domain was of binding to all three of peptides with to the affinity was observed for the in with the Fyn SH2 domain to the p + 3 specificity the Fyn SH2 domain more to the than the peptide the it a affinity for the than the peptide peptides a residue the p + that the in the and peptides play a role in binding the Fyn SH2 these three peptides in the residue the p + 2, p + 3 or p + are for the specificity for the with the Fyn SH2 domain, the variant a in affinity for the a affinity for the that a for an the p + 2 in the acquired a p + specificity. variant was to a p + specificity than Fyn SH2 domain affinity for the was to prefer p + other specificity affinity for the (p + and (p + peptides and affinity for the and that acquired a p + specificity with the Fyn SH2 Because the BG loop in is this the p + for peptide binding In variant was to have a for the p + and p + motifs than the Fyn SH2 domain binding to the peptides The in is by a could be used to the p + 3 binding and thereby p + specificity for the Intriguingly, the BG loop of a of that not the p + binding thereby the p + for on the may the p + or p + binding mode for recognition and Intriguingly, a BG for the and peptides than the Fyn SH2 with that is characterized with a BG a residue the it to p + specificity than p + the p + binding is a affinity for the than In to the variant a small residue the the same protein fold, different can recognize different The ability of to recognize a array of is in a on the of the loops the domains of I. Y. The structural of PubMed Scopus Google Scholar). These loops connect the of the and are different from one to The of recognition been in engineered from modular domains of than a typical the domain a been engineered to including that can function as an SH2 domain by the loops the S. D. proteins on the human domain 2012; PubMed Scopus (65) Google Scholar, J. S. an of the Biol. 2012; PubMed Scopus Google Scholar, J. I. J. R.B. G. S. A and highly specific of the SH2 Biol. PubMed Scopus Google Scholar, M. Z. M.D. S. recognition of that the Src Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in this the same of recognition to the SH2 we that the EF and BG loops in the Fyn SH2 domain are highly and The of the EF and BG loops the ability to encode the spectrum of specificity found in SH2 the EF and BG loops of a SH2 domain may be to acquire distinct from the domain is of loop variants selected by different bait peptides to the of Fyn SH2 that specificity from p + 3 to p + 2 or from p + 3 to p + Furthermore, we that the specificity and affinity of a variant is by the of the suggesting that the EF and BG loops not the major specificity of the SH2 domain may specificity and Although was on the Fyn SH2 domain, it is likely that other SH2 domains are of variants with a wide spectrum of specificity through loop of the EF and BG loops provides an for how different SH2 domains with the same globular structure may recognize different pY in SH2 variants with specificity may a collection of with in and cancer SH2 domains as the Fyn SH2 domain can to pY in the it to the functions of specific specificity, by a SH2 domain to an loop variant that the ability to recognize the the pY N. Huang J. M. L. L. Pawson T. S. a protein interaction in cell Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Our work that SH2 variants with specificity for a given pY may be on the SH2 by loop These variants a of pY by to tyrosine kinase signaling in Because the specificity and the are on an SH2 domain (8Kaneko T. Huang H. Cao X. Li X. Li C. Voss C. Sidhu S.S. Li S.S. Superbinder SH2 domains act as antagonists of cell signaling.Sci Signal. 2012; 5: ra68Crossref PubMed Scopus (96) Google Scholar, T. Huang H. Li L. H. Voss Wu C. Li S.S. SH2 domain specificity by to binding Signal. PubMed Scopus Google Scholar, T. Sidhu S.S. Li S.S. specificity from for protein interaction Biochem. Sci. 36: Full Text Full Text PDF PubMed Scopus Google we may be to a of SH2 variants with specificity and It be that an SH2 domain may a p + residue M. L. S. M. S. N. K. M. S. M. Mayer B.J. L. G. The SH2 domain interaction Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. G. Src SH2 domains specificity by of Biol. Full Text Full Text PDF PubMed Scopus Google and in to the (11Hwang P.M. Li C. Morra M. Lillywhite J. C. Pawson T. J.D. Li S.C. A binding mechanism for the SH2 domain: structural and to the J. PubMed Scopus Google or C-terminal to the p + M. J. Wu C. Li S.S. L. S. for by the of signaling Biol. Full Text Full Text PDF PubMed Scopus Google may not the EF or BG it can be that in of the and the specificity in an SH2 domain may a of SH2 variants with affinity and specificity for and with

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