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Phosphotyrosine Binding Domains of Shc and Insulin Receptor Substrate 1 Recognize the NPXpY Motif in a Thermodynamically Distinct Manner

1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês

10.1074/jbc.274.10.6114

1083-351X

Amjad Farooq, Olga Plotnikova, Lei Zeng, Ming‐Ming Zhou,

Ion channel regulation and function

Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites. The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors. Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites. The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors. Protein tyrosine phosphorylation provides a central control mechanism in regulating protein-protein interactions and activation of enzymes in mitogenic signal transduction following activation of cytokine and growth factor receptors (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2604) Google Scholar, 2Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2229) Google Scholar). Key events in receptor signaling are the interactions of signaling molecules such as adaptor protein Shc and insulin receptor substrate (IRS-1) 1The abbreviations used are: IRS-1, insulin receptor substrate 1; IR, insulin receptor; IL-4R, interleukin-4 receptor; ITC, isothermal titration calorimetry; NTA, nitrilotriacetic acid; pY, phosphotyrosine; PTB, phosphotyrosine binding; SASA, solvent-accessible surface area; SH2 Src homology-2, Fmoc,N-(9-fluorenyl)methoxycarbonyl; EGF, epidermal growth factor; PH, pleckstrin homology. with activated and tyrosine-phosphorylated receptors. Binding to the activated receptor results in tyrosine phosphorylation of these signaling molecules, which in turn experience specific interactions with downstream signaling proteins and/or enzymes. For example, in insulin receptor (IR) signaling, upon binding to the activated receptor, IRS-1 is phosphorylated on many tyrosine residues, which enables IRS-1 to interact with various Src homology 2 (SH2) domain-containing proteins, including phosphatidylinositol 3-kinase, protein tyrosine phosphatase SH-PTP2, and Grb2 (3Sun X.J. Crimmins D.L. Myers M.G.J. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). On the other hand, tyrosine-phosphorylated Shc interacts with the SH2 domain of the adaptor protein Grb2, which in turn binds via its Src homology 3 (SH3) domains to the guanine nucleotide exchange factor, SOS, leading to Ras activation (4Salcini A. McGlade J. Pelicci G. Nicoletti I. Pawson T. Pelicci P. Oncogene. 1994; 9: 2827-2836PubMed Google Scholar, 5Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thoma S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (827) Google Scholar). Both IRS-1 and Shc can bind to the activated and tyrosine-phosphorylated insulin receptor through their phosphotyrosine binding (PTB) domain (also called PID or SAIN domain) (6Gustafson T.A. He W. Craparo A. Schaub C.D. O'Meill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar). The PTB domain is a recently recognized protein module that can serve as an alternative to the SH2 domain for binding to tyrosine-phosphorylated proteins (6Gustafson T.A. He W. Craparo A. Schaub C.D. O'Meill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar, 7Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (451) Google Scholar, 8Blaikie P. Immanuel D. Wu J. Li N. Yajnik V. Margolis B. J. Biol. Chem. 1994; 269: 32031-32034Abstract Full Text PDF PubMed Google Scholar, 9Geer P.V.D. Wiley S. Lai K. Olivier J. Gish G. Stephens R. Kaplan D. Shoelson S. Pawson T. Curr. Biol. 1995; 5: 404-412Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). PTB domains that are structurally and functionally distinct from SH2 domains recognize amino acid residues N-terminal (rather than C-terminal) to the phosphotyrosine (pY) (2Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2229) Google Scholar, 10Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 11Zhou M.-M. Fesik S.W. Prog. Biophys. Mol. Biol. 1995; 64: 221-235Crossref PubMed Scopus (20) Google Scholar). In particular, PTB domains preferentially bind to phosphorylated proteins at sites containing a NPXpY motif and hydrophobic amino acids N-terminal to this sequence (12Trüb T. Cho W.E. Wolf G. Ottinger E. Hen Y. Weiss M. Shoelson S.E. J. Biol. Chem. 1995; 270: 18205-18208Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 13Kavanaugh W.M. Turck C.W. Williams L.T. Science. 1995; 268: 1177-1179Crossref PubMed Scopus (223) Google Scholar, 14Zhou S. Margolis B. Chaudhuri M. Shoelson S.E. Cantley L.C. J. Biol. Chem. 1995; 270: 14863-14866Crossref PubMed Scopus (159) Google Scholar, 15Wolf G. Trüb T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Unlike SH2 domains, PTB domains show very low protein sequence homology. Different PTB domains exhibit distinct selectivity for residues N-terminal to the NPXpY-motif. For example, the IRS-1 PTB domain favors hydrophobic residues at the pY-6 and pY-8 positions and an Ala at pY-1 for high affinity binding (15Wolf G. Trüb T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 16He W. O;Neill T.J. Gustafson T.A. J. Biol. Chem. 1995; 270: 23258-23262Crossref PubMed Scopus (85) Google Scholar), whereas the Shc PTB domain requires a bulky hydrophobic residue at pY-5 (12Trüb T. Cho W.E. Wolf G. Ottinger E. Hen Y. Weiss M. Shoelson S.E. J. Biol. Chem. 1995; 270: 18205-18208Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 13Kavanaugh W.M. Turck C.W. Williams L.T. Science. 1995; 268: 1177-1179Crossref PubMed Scopus (223) Google Scholar, 14Zhou S. Margolis B. Chaudhuri M. Shoelson S.E. Cantley L.C. J. Biol. Chem. 1995; 270: 14863-14866Crossref PubMed Scopus (159) Google Scholar). Recent structural analysis revealed that the two PTB domains are structurally related but employ two very different mechanisms for recognizing the phosphotyrosine and the hydrophobic residues N-terminal to the NPXpY sequence (17Zhou M.-M. Ravichandran K.S. Olejniczak E.T. Petros A.P. Meadows R.P. Sattler M. Harlan J.E. Wade W. Burakoff S.J. Fesik S.W. Nature. 1995; 378: 584-592Crossref PubMed Scopus (324) Google Scholar, 18Zhou M.-M. Huang B. Olejniczak E.T. Meadows R.P. Shuker S.B. Miyazak M. Trüb T. Shoelson S.E. Fesik S.W. Nat. Struct. Biol. 1996; 3: 388-393Crossref PubMed Scopus (107) Google Scholar, 19Eck M.J. Dhe-Pagnon S. Trüb T. Nolte R. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Indeed, except for the insulin receptor (6Gustafson T.A. He W. Craparo A. Schaub C.D. O'Meill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar), the PTB domains of Shc and IRS-1 have been shown to interact with a distinct set of growth factor and cytokine receptors. For example, the Shc PTB domain binds to activated and tyrosine-phosphorylated TrkA, ErbB2, ErbB3, and epidermal growth factor (EGF) receptors (20Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar, 21Obermeier A. Lammers R. Weismuller K. Jung G. Schlessinger J. Ullrich A. J. Biol. Chem. 1993; 268: 22963-22966Abstract Full Text PDF PubMed Google Scholar, 22Ricci A. Lanfrancone L. Chiari R. Belardo G. Pertica C. Natali P.G. Pelicci P.G. Segatto O. Oncogene. 1995; 11: 1519-1529PubMed Google Scholar), whereas IRS-1 interacts with the tyrosine-phosphorylated interleukin-4 receptor (IL-4R) via its PTB domain (23Wang L.M. Myers M.G.J. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 24Keegan A.D. Nelms K. White M. Wang L.M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Studies of thermodynamics of protein-ligand interactions can provide important insights into the structural and functional relationships of molecular recognition of the system. In an effort to determine further the structural and dynamic basis of functional differences in the molecular mechanisms by which the Shc and IRS-1 PTB domains recognize tyrosine-phosphorylated peptides, we have studied thermodynamics of peptide binding of the PTB domains using the isothermal titration calorimetry (ITC) technique. The phosphopeptides used in this study were derived from known Shc- and IRS-1-binding sites on growth factor and cytokine receptors. Results from these studies revealed that the PTB domains of Shc and IRS-1 appear to bind in a thermodynamically distinct manner to the NPXpY-containing peptides. The components of the free energy of the interactions show that the high affinity binding of the Shc PTB domain to the phosphopeptides is an overall entropy-driven process. In contrast, recognition of the IRS-1 PTB domain to the IR and IL-4R phosphopeptides is achieved predominantly by a large enthalpy contribution. By using site-directed mutagenesis and amino acid substitution, we have further quantified the relative contribution of the pY-5 and pY-1 residues in phosphopeptide binding to the PTB domains. The PTB domain of Shc (residues 17–207) was cloned, expressed, and purified using procedures as described previously (17Zhou M.-M. Ravichandran K.S. Olejniczak E.T. Petros A.P. Meadows R.P. Sattler M. Harlan J.E. Wade W. Burakoff S.J. Fesik S.W. Nature. 1995; 378: 584-592Crossref PubMed Scopus (324) Google Scholar, 25Zhou M.-M. Harlan J.E. Wade W. Crosby S. Ravichandran K.S. Burakoff S.J. Fesik S.W. J. Biol. Chem. 1995; 270: 31119-31123Crossref PubMed Scopus (49) Google Scholar). Briefly, the protein was subcloned into the bacterial expression vector pET15b (Novagen), which introduces a His tag followed by a thrombin cleavage site at the N terminus of the recombinant protein. The protein was expressed in Escherichia coli BL21(DE3) cells, which were induced with 1 mmisopropyl-1-thio-β-d-galactopyanoside for 4 h at 37 °C. The His-tagged protein was purified by affinity chromatography on a nickel-NTA column (Qiagen) and was treated with thrombin to remove the His tag. The PTB domain of IRS-1 used in this study consists of residues 157–267 of the full-length protein. A slightly larger protein (residues 157–278) was subcloned into pET30b plasmid (Novagen) and expressed in E. coli BL21(DE3pLysS) cells with an additional Leu-Glu-(His)6 sequence at the C terminus as described previously (18Zhou M.-M. Huang B. Olejniczak E.T. Meadows R.P. Shuker S.B. Miyazak M. Trüb T. Shoelson S.E. Fesik S.W. Nat. Struct. Biol. 1996; 3: 388-393Crossref PubMed Scopus (107) Google Scholar). The cells were grown overnight in LB media, and the expression of the protein was induced using 1 mmisopropyl-1-thio-β-d-galactopyanoside at 25 °C for 6 h. The cells were then disrupted using a French press. The His-tagged protein was purified by a nickel-NTA column. Subsequent cleavage of this protein with thrombin at a natural cleavage site (267–268) removed the C-terminal His-tag and the extra amino acids to give the PTB domain of IRS-1 (residues 157–267). The IRS-1 PTB mutant Met- 257 → Ala was prepared as described previously, using the Chameleon Double-Stranded, Site-directed Mutagenesis Kit (Stratagene Cloning Systems, La Jolla, CA), and the template plasmid used in the mutagenesis was pET30b-IRS1 (18Zhou M.-M. Huang B. Olejniczak E.T. Meadows R.P. Shuker S.B. Miyazak M. Trüb T. Shoelson S.E. Fesik S.W. Nat. Struct. Biol. 1996; 3: 388-393Crossref PubMed Scopus (107) Google Scholar). Expression and purification of the mutant IRS-1 PTB domain was accomplished as described for the wild-type protein. The tyrosine-phosphorylated peptides used in the experiments reported here were synthesized by the Protein Core Facility at the Mount Sinai School of Medicine, using an Fmoc-based strategy. Phosphotyrosine was incorporated using the reagent Fmoc-Tyr(PO3H2) with HBTU/HOAt activation. Analysis of the purified peptides by analytical high pressure liquid chromatography demonstrated homogeneity. Calorimetric measurements were performed with an Omega instrument (Microcal, Northampton, MA) (26Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2438) Google Scholar). All experiments were carried out at 25 °C in a 50 mm Tris-HCl buffer of pH 8.0 containing 200 mm NaCl, 5 mmβ-mercaptoethanol, and 1 mm EDTA. This condition was optimal for protein stability of the PTB domains of Shc and IRS-1, as there was no sign of significant protein aggregation for up to 0.5–1 mm protein concentration as determined by NMR spectroscopy. Both the PTB domains and the phosphopeptides were dissolved in the same buffer. The concentrations of protein and phosphopeptide were typically of 30–300 μm and 1–2 mm, respectively. To optimize the ITC measurements, the c value (c = [PTB domain]/K D) was controlled in the range of 10–200 for all the ITC experiments, except for the weak binding of the IRS-1 PTB domain to the phosphopeptides of IR-pY960 (K D = 87.07 ± 3.84 μm) and TrkA-pY490 (K D = 678 ± 96.53 μm) (Table I).Table IThermodynamic parameters obtained for binding of the Shc PTB domain to NPXpY-containing phosphopeptides at pH 8.0 and 25 °CProtein pY sitespY peptidesK DΔHTΔSΔGμmkcal mol−1kcal mol−1kcal mol−1TrkA, pY490HIIENPQpYFSDA0.19 ± 0.013.63 ± 0.1112.75 ± 0.15−9.12 ± 0.03hEGFR, pY1148SLDNPDpYQQDFF1.69 ± 0.15−2.82 ± 0.085.02 ± 0.11−7.84 ± 0.05hErbB3, pY1309SAFDNPDpYWHSRLF0.33 ± 0.04−2.54 ± 0.086.27 ± 0.13−8.81 ± 0.08IR, pY960LYASSNPEpYLS4.22 ± 0.902.54 ± 0.299.85 ± 0.16−7.31 ± 0.14IL-4R, pY497LVIAGNPApYRS12.25 ± 5.220.87 ± 0.147.58 ± 0.15−6.71 ± 0.28TrkA A-5, pY490HIAENPQpYFSDA0.70 ± 0.172.78 ± 0.1411.16 ± 0.10−8.38 ± 0.14The experimental conditions of the ITC measurements were described in detail under "Experimental Procedures." Three ITC experiments were conducted for each phosphopeptide at slightly different protein concentrations. The values for K D(K D = I/K B) and ΔHwere calculated directly from the curve fitting of the titration data to a function based on the binding of a ligand to a macromolecule (22Ricci A. Lanfrancone L. Chiari R. Belardo G. Pertica C. Natali P.G. Pelicci P.G. Segatto O. Oncogene. 1995; 11: 1519-1529PubMed Google Scholar), using ORIGIN. In this fitting procedure, the values forK B, ΔH, and n (reaction stoichiometry) were all allowed to float. The mean value forn was found to be 1 ± 0.1. Errors quoted forK D and ΔH are standard deviations from the three ITC experiments, whereas errors onTΔS and ΔG are propagated errors. Open table in a new tab The experimental conditions of the ITC measurements were described in detail under "Experimental Procedures." Three ITC experiments were conducted for each phosphopeptide at slightly different protein concentrations. The values for K D(K D = I/K B) and ΔHwere calculated directly from the curve fitting of the titration data to a function based on the binding of a ligand to a macromolecule (22Ricci A. Lanfrancone L. Chiari R. Belardo G. Pertica C. Natali P.G. Pelicci P.G. Segatto O. Oncogene. 1995; 11: 1519-1529PubMed Google Scholar), using ORIGIN. In this fitting procedure, the values forK B, ΔH, and n (reaction stoichiometry) were all allowed to float. The mean value forn was found to be 1 ± 0.1. Errors quoted forK D and ΔH are standard deviations from the three ITC experiments, whereas errors onTΔS and ΔG are propagated errors. Each titration experiment consisted of 25 10-μl injections of a peptide into the calorimetric cell containing 1.34 ml of a protein solution. A 250-s period was allowed between each injection, and there was an initial 60-s delay at the start of the experiment. Reaction enthalpies were also measured for injection of buffer into the protein and the phosphopeptide into the buffer. In each case, the measured enthalpies were found to be negligible compared with the enthalpy of the binding of the phosphopeptide to the PTB domains. The mean of the enthalpy of injection of buffer into the protein was subtracted from raw titration data prior to curve fitting. The peptide concentration was determined gravimetrically, whereas the protein concentration was measured using the Lowry method. Titration curves were fit to an in-built function by a non-linear least squares method using the ORIGIN software (Microcal, Northampton, MA). This function is based upon the binding of a ligand to a macromolecule (26Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2438) Google Scholar) and contains n(reaction stoichiometry), K D (dissociation constant), and ΔH (reaction enthalpy) as the variable parameters. These parameters can thus be directly determined from curve fitting. From the values of K D and ΔH, the free energy (ΔG) and entropy change (ΔS) upon peptide binding can be calculated using the relationship: −RT ln(1/K D) = ΔG = ΔH − TΔS, where Ris the universal molar gas constant and T is the absolute temperature. All NMR spectra were acquired at 30 °C on a Bruker DRX-500 NMR spectrometer. Uniformly 15N-labeled proteins of the IRS-1 PTB domain were prepared for the NMR experiments by growing bacteria that overexpress the PTB domain in an M9 minimal medium containing 15NH4Cl as the sole nitrogen source. The NMR samples of wild-type and the Met-257 → Ala mutant of the IRS-1 PTB domain were prepared at a concentration of 0.5 mm in 50 mm Tris-d 11/HCl buffer of pH 6.5, containing 50 mm NaCl and 5 mm dithiothreitol-d 10 in 90% H2O, 10% 2H2O. Two-dimensional1H/15N heteronuclear single quantum coherence spectra were acquired with 96 and 1024 complex points in ω1 and ω2, respectively. The NMR spectra were processed and analyzed using the NMRPipe (27Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar) and NMRView (28Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2678) Google Scholar) programs. We used the same TrkA receptor peptide (HIIENPQpYFSDA) in the isothermal titration calorimetry studies as the one used in our recent structural analysis of the Shc PTB domain-TrkA phosphopeptide complex by NMR (17Zhou M.-M. Ravichandran K.S. Olejniczak E.T. Petros A.P. Meadows R.P. Sattler M. Harlan J.E. Wade W. Burakoff S.J. Fesik S.W. Nature. 1995; 378: 584-592Crossref PubMed Scopus (324) Google Scholar). From a peptide titration experiment using ITC, one can obtain thermodynamic information of the binding process (29Livingstone J.R. Nature. 1996; 384: 491-492Crossref PubMed Scopus (22) Google Scholar, 30Ladbury J.E. Lemmon M.A. Zhou M. Green J. Botfield M.C. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3199-3203Crossref PubMed Scopus (243) Google Scholar). The parameters include binding affinity, binding stoichiometry, enthalpy of binding (ΔH), and free energy change (ΔG) by a nonlinear fit of the binding isotherm, as well as entropy of binding (ΔS) from a difference between the free-energy change and the enthalpy of binding. A representative calorimetric isotherm and the corresponding titration curve of the Shc PTB domain binding to the TrkA peptide (Fig. 1 A) show that a heat absorbance is associated with the peptide binding, indicating that the interaction is endothermic at 25 °C (ΔH = 3.63 kcal/mol). The heat absorbance upon addition of the phosphopeptide to the protein solution underwent a sharp change at 1:1 molar ratio of the protein to peptide, suggesting that the Shc PTB domain binding to the TrkA peptide is very tight, and the stoichiometry of this interaction is 1:1. By using these ITC data, we calculated a dissociation constant (K D) to be 190 nm (Table I). Furthermore, the thermodynamic titration data revealed that the high affinity binding of the Shc PTB domain to the TrkA peptide is achieved by an overall entropy-driven process as the free energy of binding (ΔG = −9.12 kcal/mol) results predominantly from a large favorable entropic contribution (TΔS= 12.75 kcal/mol).Figure 1Isothermal titration calorimetric data for binding of the Shc PTB domain to TrkA-pY490 (A) and the IRS-1 PTB domain to tyrosine-phosphorylated peptides of IL4R-pY497 (B), IR-pY960 (C), and IR(A-1)-pY960 (D). The solid lines show the fit of the data to a function based on the binding of a ligand to a macromolecule using the software ORIGIN (26Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2438) Google Scholar).View Large Image Figure ViewerDownload (PPT) To determine how the Shc PTB domain interacts thermodynamically with other NPXpY-containing phosphopeptides, we measured thermodynamic parameters of the Shc PTB domain binding to tyrosine-phosphorylated peptides derived from EGF, ErbB3, and insulin receptors (Table I). The results indicated that while the enthalpy of binding (either exothermic or endothermic reaction) is phosphopeptide-specific, change of entropy (TΔS) always favors the binding. Moreover, this large favorable entropic contribution appears to be the major determinant for the high affinity of the Shc PTB domain binding to the phosphopeptides. This observation is consistent not only with the phosphopeptides that contain the consensus sequence of ΨXNPXpY (where Ψ pY-5 is a hydrophobic residue) known for the high affinity binding to the Shc PTB domain but also with the IR phosphopeptide that contains large hydrophobic residues at pY-6 to pY-8 instead of pY-5. To study further the thermodynamics of the PTB domain of Shc binding to tyrosine-phosphorylated peptides, we conducted ITC measurements using a phosphopeptide derived from IL-4 receptor (pY497). This IL-4R phosphopeptide is not a biological ligand for the Shc PTB domain as Shc has not been linked to IL-4R signaling. On the other hand, the IL-4R peptide represents a biologically relevant binding site for the IRS-1 PTB domain (23Wang L.M. Myers M.G.J. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 24Keegan A.D. Nelms K. White M. Wang L.M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar). The ITC results showed that the PTB domain of Shc binds to the IL-4R peptide much weaker than to those phosphopeptides from the known Shc binding sites (Table I). Interestingly, the Shc binding of the IL-4R peptide is also dictated by a large favorable entropic contribution. Taken together, our ITC results suggest that under the conditions of our study, binding of the Shc PTB domain to the NPXpY-containing phosphopeptides of the TrkA, EGF, ErbB3, and insulin receptors appears to be an overall entropy-driven process. We performed an ITC titration using a phosphopeptide from the IL-4R (pY497) (LVIAGNPApYRS) which is a known binding site for IRS-1 (23Wang L.M. Myers M.G.J. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 24Keegan A.D. Nelms K. White M. Wang L.M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar). As shown in Fig. 1 B, binding of the IRS-1 PTB domain to the IL-4R peptide (see Table II) is exothermic (ΔH = −9.43 kcal/mol) and involves an unfavorable change of entropy (TΔS = −1.63 kcal/mol). Curve fitting of the ITC data gave a dissociation constant K D of 1.82 μm for this PTB domain-peptide complex. Thus, the IL-4R peptide interaction with the IRS-1 PTB domain is enthalpy-driven, which is in sharp contrast to the entropy-driven binding of the Shc PTB domain.Table IIThermodynamic parameters obtained for binding of the IRS-1 PTB domain to NPXpY-containing phosphopeptides at pH 8.0 and 25 °CProtein pY sitespY peptidesK DΔHTΔSΔGμmkcal mol−1kcal mol−1kcal mol−1IL-4R, pY497LVIAGNPApYRS1.82 ± 0.11−9.43 ± 0.15−1.63 ± 0.17−7.80 ± 0.04IR, pY960LYASSNPEpYLS87.07 ± 3.84−10.74 ± 0.30−5.22 ± 0.33−5.52 ± 0.03IR (A-1), pY960LYASSNP A pYLS2.32 ± 0.25−10.85 ± 0.09−3.19 ± 0.15−7.66 ± 0.07IR (I-1), pY960LYASSNP I pYLS8.40 ± 1.14−5.91 ± 0.110.99 ± 0.18−6.90 ± 0.08IR (F-1), pY960LYASSNP F pYLS6.64 ± 0.27−5.35 ± 0.071.69 ± 0.09−7.04 ± 0.03IR (A-1), Y960LYASSNPAYLS∼0aThese thermodynamic parameters cannot be determined since the measured signal (ΔH) is observed to be zero.TrkA, pY490HIIENPQpYFSDA678.02 ± 96.53−9.65 ± 0.76−5.34 ± 0.85−4.31 ± 0.08hEGFR, pY1148SLDNPDpYQQDFF∼0aThese thermodynamic parameters cannot be determined since the measured signal (ΔH) is observed to be zero.hErbB3, pY1309SAFDNPDpYWHSRLF∼0aThese thermodynamic parameters cannot be determined since the measured signal (ΔH) is observed to be zero.IRS-1 PTB-M257AIL-4R, pY497LVIAGNPApYRS24.64 ± 2.21−8.36 ± 0.37−2.10 ± 0.42−6.26 ± 0.06IR (A-1), pY960LYASSNP A pYLS21.98 ± 0.22−7.58 ± 0.08−1.25 ± 0.07−6.33 ± 0.02The experimental conditions of the ITC measurements were described in detail under "Experimental Procedures" and Table I. The mean value for n (reaction stoichiometry) was found to be 1 ± 0.1. Errors quoted for K D and ΔH are standard deviations from the three ITC experiments, whereas errors onTΔS and ΔG are propagated errors.a These thermodynamic parameters cannot be determined since the measured signal (ΔH) is observed to be zero. Open table in a new tab The experimental conditions of the ITC measurements were described in detail under "Experimental Procedures" and Table I. The mean value for n (reaction stoichiometry) was found to be 1 ± 0.1. Errors quoted for K D and ΔH are standard deviations from the three ITC experiments, whereas errors onTΔS and ΔG are propagated errors. Binding of the IRS-1 PTB domain to the IR-pY960 peptide (LYASSNPEpYLS) also appears to be governed mainly by an enthalpy contribution (ΔH = −10.74 kcal/mol andTΔS = −5.22 kcal/mol). However, binding affinity of the IR-pY960 peptide to the IRS-1 PTB domain (K D = 87.07 μm) is ∼50-fold weaker than that of the IL4R-pY497 peptide (K D = 1.82 μm) (Fig. 1 C, Table II). This marked reduction of the peptide binding affinity correlates with an increased entropy penalty. The major difference in amino acid sequence between the IL-4R and IR phosphopeptides is the residue at pY-1. To determine the contribution of the pY-1 residue to binding of the IRS-1 PTB domain, we substituted the Glu pY-1 in the IR-pY960 peptide by an Ala. The latter amino acid corresponds to the Ala pY-1 in the IL-4R phosphopeptide. The ITC measurements showed that this single amino acid substitution led to a 38-fold increase of the binding affinity to 2.32 μm (Fig.1 D and Table II), which is nearly the same as that of the IL-4R peptide binding to the IRS-1 PTB domain (K D = 1.82 μm). It is interesting to note that th