Structural Requirements for Signal Transducer and Activator of Transcription 3 Binding to Phosphotyrosine Ligands Containing the YXXQ Motif
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m314037200
ISSN1083-351X
AutoresHuang Shao, Xuejun Xu, Mary-Ann A. Mastrangelo, Naijie Jing, Richard G. Cook, Glen B. Legge, David J. Tweardy,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoStat3 is an Src homology (SH)2-containing protein constitutively activated in a wide variety of human cancers following its recruitment to YXXQ-containing motifs, which results in resistance to apoptosis. Despite resolution of the crystal structure of Stat3 homodimer bound to DNA, the structural basis for the unique specificity of Stat3 SH2 for YXXQ-containing phosphopeptides remains unresolved. We tested three models of this interaction based on computational analysis of available structures and sequence alignments, two of which assumed an extended peptide configuration and one in which the peptide had a β-turn. By using peptide immunoblot affinity assays and mirror resonance affinity analysis, we demonstrated that only phosphotyrosine (Tyr(P)) peptides containing +3 Gln (not Leu, Met, Glu, or Arg) bound to wild type Stat3. Examination of a series of wild type and mutant Stat3 proteins demonstrated loss of binding to pYXXQ-containing peptides only in Stat3 mutated at Lys-591 or Arg-609, whose side chains interact with the Tyr(P) residue, and Stat3 mutated at Glu-638, whose amide hydrogen bonds with oxygen within the +3 Gln side chain when the peptide ligand assumes a β-turn. These findings support a model for Stat3 SH2 interactions that could form the basis for anticancer drugs that specifically target Stat3. Stat3 is an Src homology (SH)2-containing protein constitutively activated in a wide variety of human cancers following its recruitment to YXXQ-containing motifs, which results in resistance to apoptosis. Despite resolution of the crystal structure of Stat3 homodimer bound to DNA, the structural basis for the unique specificity of Stat3 SH2 for YXXQ-containing phosphopeptides remains unresolved. We tested three models of this interaction based on computational analysis of available structures and sequence alignments, two of which assumed an extended peptide configuration and one in which the peptide had a β-turn. By using peptide immunoblot affinity assays and mirror resonance affinity analysis, we demonstrated that only phosphotyrosine (Tyr(P)) peptides containing +3 Gln (not Leu, Met, Glu, or Arg) bound to wild type Stat3. Examination of a series of wild type and mutant Stat3 proteins demonstrated loss of binding to pYXXQ-containing peptides only in Stat3 mutated at Lys-591 or Arg-609, whose side chains interact with the Tyr(P) residue, and Stat3 mutated at Glu-638, whose amide hydrogen bonds with oxygen within the +3 Gln side chain when the peptide ligand assumes a β-turn. These findings support a model for Stat3 SH2 interactions that could form the basis for anticancer drugs that specifically target Stat3. Signal transducer and activator of transcription 3 is a latent transcription factor activated by cytokine and growth factor receptors including interleukin-6 and EGFR 1The abbreviations used are: EGFR, epidermal growth factor receptor; SH, Src homology; mAb, monoclonal antibody; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; PDP, phosphododecapeptide. 1The abbreviations used are: EGFR, epidermal growth factor receptor; SH, Src homology; mAb, monoclonal antibody; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; PDP, phosphododecapeptide. (1Wegenka U.M. Buschmann J. Lutticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Google Scholar, 2Akira S. Nishio Y. Inoue M. Wang X.J. Wei S. Matsusaka T. Yoshida K. Sudo T. Naruto M. Kishimoto T. Cell. 1994; 77: 63-71Google Scholar, 3Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Google Scholar). Stat3 is recruited to the cytoplasmic domain of receptors via its SH2 domain and phosphorylated on tyrosine 705 by either intrinsic or receptor-associated tyrosine kinases, most notably members of the Janus family. Phosphorylation of Stat3 leads to dimerization mediated by reciprocal SH2-Tyr(P)-705 motif interactions, followed by nuclear translocation, binding to specific DNA elements, and up-regulation of target genes.Stat3 has been demonstrated to be required for transformation of fibroblasts by v-Src (4Turkson J. Bowman T. Garcia R. Caldenhoven R. DeGroot R.P. Jove R. Mol. Cell. Biol. 1998; 18: 2545-2552Google Scholar, 5Bromberg J.F. Horvath C.M. Besser D. Lathem W.W. Darnell Jr., J.E. Mol. Cell. Biol. 1998; 18: 2553-2558Google Scholar) and for autocrine growth of squamous cell carcinoma of the head and neck (6Grandis J.R. Drenning S.D. Chakraborty A. Zhou M.Y. Zeng Q. Pitt A.S. Tweardy D.J. J. Clin. Investig. 1998; 102: 1385-1392Google Scholar) where it is activated by an autocrine loop involving TGF-β and EGFR (7Grandis J.R. Tweardy D.J. Cancer Res. 1993; 53: 3579-3584Google Scholar). Expression of a constitutively activated form of Stat3 alone in fibroblasts was oncogenic (8Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Google Scholar). Constitutive activation of Stat3 occurs in a wide variety of cancers in addition to squamous cell carcinoma of the head and neck including breast, prostate, renal cell, melanoma, ovarian, lung, leukemia, lymphoma, and multiple myeloma (9Bowman T. Garcia R. Turkson J. Jove R. Oncogene. 2000; 19: 2474-2488Google Scholar) as a result of autocrine or paracrine activation of the EGFR and the interleukin-6 receptor or secondary to as yet unidentified mechanisms.EGFR contains an extracellular ligand-binding domain, a single transmembrane region, and an intracellular domain harboring intrinsic tyrosine kinase activity (10Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. et al.Nature. 1984; 309: 418-425Google Scholar). Ligand-induced dimerization of EGFR allows reciprocal transphosphorylation of residues within the catalytic domain of the kinase leading to its enzymatic activation and autophosphorylation of C-terminal cytoplasmic tyrosine residues. Five autophosphorylation sites have been identified in EGFR as follows: Tyr-992, Tyr-1068, Tyr-1086, Tyr-1148, and Tyr-1173 (11Downward J. Parker P. Waterfield M.D. Nature. 1984; 311: 483-485Google Scholar, 12Margolis B. Li N. Koch A. Mohammadi M. Hurwitz D.R. Zilberstein A. Ullrich A. Pawson T. Schlessinger J. EMBO J. 1990; 9: 4375-4380Google Scholar). These phosphorylated tyrosine residues serve as docking sites for signal proteins containing Src homology (SH2) domains, including phospholipase C-γ (13Rotin D. Margolis B. Mohammadi M. Daly R.J. Daum G. Li N. Fischer E.H. Burgess W.H. Ullrich A. Schlessinger J. EMBO J. 1992; 11: 559-567Google Scholar, 14Chattopadhyay A. Vecchi M. Ji Q. Mernaugh R. Carpenter G. J. Biol. Chem. 1999; 274: 26091-26097Google Scholar), Grb-2 (15Okutani T. Okabayashi Y. Kido Y. Sugimoto Y. Sakaguchi K. Matuoka K. Takenawa T. Kasuga M. J. Biol. Chem. 1994; 269: 31310-31314Google Scholar, 16Batzer A.G. Rotin D. Urena J.M. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Google Scholar), Shc (17Okabayashi Y. Kido Y. Okutani T. Sugimoto Y. Sakaguchi K. Kasuga M. J. Biol. Chem. 1994; 269: 18674-18678Google Scholar), SHP-1 (18Keilhack H. Tenev T. Nyakatura E. Godovac-Zimmermann J. Nielsen L. Seedorf K. Bohmer F.D. J. Biol. Chem. 1998; 273: 24839-24846Google Scholar), and most recently Stat3 (19Shao H. Cheng H.Y. Cook R.G. Tweardy D.J. Cancer Res. 2003; 63: 3923-3930Google Scholar), which was shown by us to bind to EGFR at Tyr(P) sites located at Tyr-1068 and Tyr-1086. Both of these tyrosine residues are followed at the Tyr(P) +3 position by Gln thereby conforming to the consensus Stat3 SH2-binding motif, YXXQ (20Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Google Scholar, 21Weber-Nordt R.M. Riley J.K. Greenlund A.C. Moore K.W. Darnell J.E. Schreiber R.D. J. Biol. Chem. 1996; 271: 27954-27961Google Scholar). The preference of Stat3 SH2 for Tyr(P) peptide ligands containing Gln (or the polar residues Thr or Cys) at the +3 position is unique among SH2 domains. The structural basis for this is unknown but could be exploited to specifically target Stat3 activation in cancers such as squamous cell carcinoma of the head and neck in which Stat3 activation occurs downstream of activated EGFR.Although the structure of Stat3 SH2 bound to Tyr(P) ligand has not been solved, the structure of Stat3β bound to DNA has been encompassing the domains of Stat3β from residues 127 to 722 including the SH2 domain (22Becker S. Groner B. Muller C.W. Nature. 1998; 394: 145-151Google Scholar). The authors concluded that Stat3 SH2 shares structural features of other SH2 domains having a central, three-stranded anti-parallel β-pleated sheet (strands B-D) flanked by helix αA and strands βA and βG. However, since the electron density was not well defined for the SH2 domain and the Tyr(P)705-containing phosphopeptide region, the structure obtained did not clarify the preference of Stat3 SH2 for binding to phosphopeptide ligands with Tyr(P) +3 Gln (or +3 Thr because Thr-708 is located at the +3 position downstream of Tyr(P)-705). Two models have been proposed to explain this preference (23Hemmann U. Gerhartz C. Heesel B. Sasse J. Kurapkat G. Grotzinger J. Wollmer A. Zhong Z. Darnell Jr., J.E. Graeve L. Heinrich P.C. Horn F. J. Biol. Chem. 1996; 271: 12999-13007Google Scholar, 24Chakraborty A. Dyer K.F. Cascio M. Mietzner T.A. Tweardy D.J. Blood. 1999; 93: 15-24Google Scholar); both assume an extended configuration for the Tyr(P) peptide ligand and two pockets as follows: one, a positively charged pocket that interacts with the Tyr(P) residue; and the other, a hydrophilic pocket that interacts with the +3 Gln; but neither model has been tested and verified.By using wild type and mutated EGFR Tyr-1068 PDPs in peptide immunoblot and mirror resonance affinity analyses, we demonstrated the following. 1) Tyr(P) binding requires interaction of the phosphate group with the side chains of Lys-591 and Arg-609 within the Stat3 SH2. 2) The +3 Gln is required for binding of Stat3 to pYXXQ-containing peptides. 3) Binding of Stat3 SH2 to pYXXQ-containing peptides does not require the side chains of Glu-638, Tyr-640, and Tyr-657 or Tyr-657, Cys-687, Ser-691, and Glu-692 proposed to form pocket 2 in the Chakraborty et al. (24Chakraborty A. Dyer K.F. Cascio M. Mietzner T.A. Tweardy D.J. Blood. 1999; 93: 15-24Google Scholar) and Hemmann et al. (23Hemmann U. Gerhartz C. Heesel B. Sasse J. Kurapkat G. Grotzinger J. Wollmer A. Zhong Z. Darnell Jr., J.E. Graeve L. Heinrich P.C. Horn F. J. Biol. Chem. 1996; 271: 12999-13007Google Scholar) models, respectively. Rather, our affinity analysis coupled with computer modeling supports a model in which the Tyr(P) ligand has a β-turn and the oxygen on the side chain of the +3 Gln forms a bond with the amide hydrogen within the peptide backbone of Stat3 at Glu-638. These findings have important implications for design of peptidomimetics to specifically target Stat3 recruitment and activation in cancer cells.EXPERIMENT PROCEDURESSite-directed Mutagenesis of Stat3 and EGFR—Human Stat3α cDNA was a gift from Dr. Rolf Van de Groot (25Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A.M. Lammers J.W.J. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Google Scholar). A HindIII/XhoI DNA fragment containing Stat3α was cloned into the baculovirus expression vector, pFastBac1 (Invitrogen), with a 6-histidine tag engineered onto the N terminus of human Stat3. Single or combination mutations were generated by using Quikchange site-directed mutagenesis kit (Stratagene) to target amino acid residues within the Stat3 SH2 domain implicated in models of Stat3 SH2-phosphotyrosine binding (K591L, R609L, E638P, E638L, Y640F, Y657F, C687A, S691A, and Q692L; Fig. 1). The sequence of each constructs was verified by sequencing analysis.Expression and Purification of Stat3 Proteins—The wild type and mutated Stat3 plasmid was used to transform DH10Bac-competent cells, which contain a bacmid with a mini-attTn7 target site and helper plasmid. Recombinant bacmids were prepared and used to infect Sf9 cells. Sf9 cells (3 × 106 cells per ml) were infected with Stat3 recombinant virus at a multiplicity of infection of 0.05 and harvested after a 3-day culture. Cells (6 × 108) were suspended in 12 ml of pre-cooled lysis buffer (20 mm Tris-Cl, pH 8.0, 0.5 m NaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 10 mm imidazole) and lysed by ultrasonication on ice. Lysates were centrifuged at 15,000 × g for 30 min at 4 °C, and the supernatant was incubated with Ni-NTA-agarose (Qiagen) at 4 °C for 1 h. The Ni-NTA resin was washed twice with 4 volumes of wash buffer (20 mm Tris-Cl, pH 8.0, 0.5 m NaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 20 mm imidazole) to remove unbound proteins. Stat3 was eluted from the Ni-NTA resin with elution buffer (20 mm Tris-Cl, pH 8.0, 0.5 m NaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 250 mm imidazole). The purified proteins were dialyzed against 10 mm PBS at 4 °C and stored at -80 °C.Peptide Synthesis—The peptides listed in Table I were synthesized in the Baylor College of Medicine Protein Core Facility on an Applied Biosystems (Foster City, CA) model 433A peptide synthesizer using standard 9-fluorenylmethoxycarbonyl amino acid chemistry. Seventy percent of the peptide reaction mix was biotinylated at the N terminus while the peptide remained on the resin using d-Biotin-LC (AnaSpec, Inc.). All peptides were purified using reverse-phase high performance liquid chromatography and were ≥95% pure.Table ITyrosine-phosphorylated and non-phosphorylated peptides synthesized based upon the EGFR sequencePeptideSequenceTyr(P)-992TDSNF(pY)RALMDETyr(P)-1068LPVPE(pY)INQSVPpY1068RLPVPE(pY)INRSVPpY1068ELPVPE(pY)INESVPpY1068MLPVPE(pY)INMSVPpY1068LLPVPE(pY)INLSVPTyr-1068LPVPEYINQSVPTyr(P)-1086VQNPV(pY)HNQPLNTyr-1086VQNPVYHNQPLNTyr(P)-1148VGNPE(pY)LNTVQPTyr(P)-1173LDNPD(pY)QQDFFP Open table in a new tab Phosphopeptide Affinity Immunoblot Analysis—NeutrAvidin-agarose (40 μl; Pierce) was incubated with 10 μg of biotinylated peptide in 300 μl of Buffer A (20 mm HEPES, pH 7.5, 20 mm NaF, 1 mm Na3VO4, 1 mm Na4P2O7, 1 mm EDTA, 1 mm EGTA, 20% glycerol, 0.05% Nonidet P-40, 1 mm dithiothreitol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, 100 mm NaCl) at 4 °C for 2 h and washed with Buffer A three times. The NeutrAvidin-peptide complex was then mixed with His-tagged Stat3 protein (5 μg) in 1 ml of Buffer A (without NaCl and Nonidet P-40) at 4 °C for 2 h and washed thoroughly. Bound proteins were separated and immunoblotted using Stat3 monoclonal antibody (mAb) as described (19Shao H. Cheng H.Y. Cook R.G. Tweardy D.J. Cancer Res. 2003; 63: 3923-3930Google Scholar).Mirror Resonance Affinity Assay—Kinetics experiments were performed using an Iasys Auto+ resonant mirror biosensor (Affinity Sensor, Paramus, NJ) as described (26Schuenke K.W. Cook R.G. Rich R.R. Hum. Immunol. 1998; 59: 783-793Google Scholar). Briefly, two-welled cuvettes coated on the bottom of each well with biotin were purchased from Affinity Sensor and prepared for immobilization of biotinylated peptides by coating each surface with 0.04 mg/ml NeutrAvidin (Pierce) and washing with PBS-T (20 mm sodium phosphate, 0.05% Tween 20). Biotinylated peptide (5 μg) was added into each well, experimental peptide to one well and control peptide to the other, and change in arc seconds was monitored simultaneously in both wells by using the biosensor until stable followed by washing with PBS-T. Real time binding of Stat3 was conducted at 25 °C at a stir speed of 70 for 10 min starting at the lowest concentration of Stat3. The wells were washed out with three changes of 60 μl of PBS-T, and dissociation was allowed to proceed for 5 min. Each well bottom was regenerated by washing with 50 μl of 100 mm formic acid for 2 min and equilibrated with PBS-T for the next round of association assay. Data were collected automatically and analyzed with the FASTplot and GraFit software (27Leatherbarrow R.J. GraFit. Erithacus Software Ltd., Staines1998Google Scholar).CD—CD spectra of the wild type and E638P mutants of Stat3 were recorded between the 280 and 190 nm range in 10 mm phosphate-buffered saline on an Olis DSM 1000 CD spectrophotometer. Measurements were performed at a protein concentration of 1.8 and 1.6 μm for the wild type and mutant Stat3, respectively, using a 1-mm cuvette. Spectra were acquired at 10 °C with a 2-s integration time and repeated three times for each sample.RESULTSRequirement for +3 Glu within the Tyr-1068 Phosphopeptide Ligand for Stat3 Binding—Our previous studies indicated that sites of autophosphorylation within the C terminus of EGFR at Tyr-1068 and Tyr-1086, which are each followed at the Tyr(P) +3 position by Gln, mediated the recruitment of Stat3 leading to its activation (19Shao H. Cheng H.Y. Cook R.G. Tweardy D.J. Cancer Res. 2003; 63: 3923-3930Google Scholar). Peptide affinity immunoblot analysis and mirror resonance imaging studies using phosphorylated and non-phosphorylated dodecapeptides based on the amino acid sequence within the region of the EGFR containing Tyr-1068 and Tyr-1086 demonstrated the requirement for their phosphorylation on tyrosine to achieve measurable binding of native and recombinant Stat3. These studies also revealed that Tyr-1068 phosphododecapeptide bound with 2-fold higher affinity than Tyr-1086 PDP.To determine whether or not the polar residue Gln at the +3 position of Tyr(P)-1068 peptide is essential for Stat3 SH2 binding, we synthesized a panel of tyrosine-phosphorylated dodecapeptides based on Tyr-1068 in which +3 Gln was left unchanged or replaced by a residue with a non-polar side chain Leu or Met, an acidic side chain Glu, or a basic side chain Arg (Table I). Each peptide was incubated with equal amounts of purified wild type Stat3 protein in peptide pull-down assays (Fig. 1). Immunoblotting for Stat3 demonstrated a prominent Stat3 band in pull-down assays using wild type Tyr-1068 PDP. In contrast, little to no Stat3 was detected in pull-down assays using PDPs in which the Gln was mutated to Leu, Met, Glu, or Arg similar to results using unphosphorylated Tyr-1068 dodecapeptide. Thus, Gln at the +3 position of Tyr-1068 phosphopeptide is required for Stat3 binding and appears to be as important for Stat3 binding as phosphorylation on tyrosine. Real time resonance mirror affinity assays using Tyr-1068 Gln to Arg PDP, which was the only PDP to demonstrate any detectable binding of Stat3 in peptide immunoblot studies, demonstrated that mutation of Gln to Arg decreased Stat3 binding to undetectable levels (data not shown).The Side Chains of Lys-591 and Arg-609 within Pocket 1 of Stat3, but Not the Side Chains of Amino Acid Residues within Pocket 2, Are Essential for Stat3 Binding to YXXQ-containing Phosphopeptides—Hemmann et al. (23Hemmann U. Gerhartz C. Heesel B. Sasse J. Kurapkat G. Grotzinger J. Wollmer A. Zhong Z. Darnell Jr., J.E. Graeve L. Heinrich P.C. Horn F. J. Biol. Chem. 1996; 271: 12999-13007Google Scholar) and we (24Chakraborty A. Dyer K.F. Cascio M. Mietzner T.A. Tweardy D.J. Blood. 1999; 93: 15-24Google Scholar) proposed previously two distinct but overlapping two-pocket models for the binding of YXXQ-containing PDP ligands by the Stat3 SH2 domain; both models assumed the peptide ligand was in an extended configuration (Fig. 2, A and B). In our model, the phosphotyrosine residue interacts with a positively charged pocket (pocket 1) within the SH2 domain formed primarily by the side chains of Lys-591 and Arg-609 and secondarily by the side chains of Ser-611, Glu-612, and Ser-613. The Tyr(P) +3 Gln was predicted to interact with a hydrophilic pocket (pocket 2) formed by the side chains of Glu-638, Tyr-640, and Tyr-657. In the Hemmann model, the phosphotyrosine was predicted to interact with the side chain of Arg-609 (pocket 1) and the +3 Gln with the side chains of Tyr-657, Cys-687, Ser-691, and Glu-692 (pocket 2).Fig. 2Models of Stat3 SH2-phosphotyrosine binding and Stat3 proteins generated to test them. Schematic representation of the two models of Stat3 SH2 binding to pYXXQ peptide proposed by Chakraborty (A) and Hemmann (B) each involving two pockets. The phosphotyrosine (pY) interacts with a positively charged pocket formed by the side chains of Lys-591, Arg-609, Ser-611, Glu-612, and Ser-613 (A) or by Arg-609 (B), whereas Tyr(P) +3 Gln interacts with a hydrophilic pocket within the SH2 domain formed by the side chains of Glu-638, Tyr-640 and Tyr-657 (A) or Tyr-657, Cys-687, Ser-691, and Glu-692 (B). C, mutations were introduced at the amino acid residues indicated (+) to generate a panel of wild type and mutant Stat3 proteins. D, wild type and mutant Stat3 proteins, each with an N-terminal His tag, were expressed in Sf9 insect cells and affinity-purified using Ni-NTA-agarose. The eluates were separated by SDS-PAGE, and the gel was stained with Coomassie Blue (top panel) or immunoblotted using Stat3 mAb (bottom panel).View Large Image Figure ViewerDownload (PPT)In order to test each of the two models proposed, we generated Stat3 mutants in which mutations were introduced to change charged or polar side chains to non-polar within amino acid residues predicted in each model to be critical for Stat3 binding (Fig. 2C). His tags were added at the N terminus of each protein to aid in purification; we demonstrated previously that this modification did not interfere with binding of wild type Stat3 to native full-length, activated EGFR, or to PDPs Tyr-1068 and Tyr-1086. The recombinant Stat3 proteins were expressed in Sf9 insect cells and purified to equivalent levels using Ni-NTA resin (Fig. 2D).Peptide affinity immunoblot studies using Stat3-3M to test the pocket 2 component of the Chakraborty model demonstrated levels of Stat3-3M bound to Tyr-1068 and Tyr-1086 PDPs similar to wild type Stat3 (Fig. 3A). Peptide affinity immunoblot studies using Stat3-4M to test the pocket 2 component of the Hemmann model demonstrated levels of binding of Stat3-4M bound to Tyr-1068 and Tyr-1086 phosphopeptides equal to or greater than wild type Stat3 (Fig. 3A). Stat3-6M, in which all six amino acid residues predicted by both models to form pocket 2 were mutated, also bound both PDPs at levels similar to wild type Stat3 as did Stat3-2M and Stat3-3M+C687A. These results do not support either model for Stat3 SH2 binding to +3 Glu within phosphopeptide ligands.Fig. 3Requirement for Arg-609 and Lys-591, but not any of the proposed pocket 2 residues, for Stat3 SH2 binding to Tyr-1068 and Tyr-1086 PDP. A, NeutrAvidin-agarose was incubated with the indicated biotinylated peptides (see Table I for sequence) or no peptide (CON) as control, washed thoroughly, and mixed with identical amounts of wild type or mutant Stat3 proteins as indicated. Bound proteins were separated by SDS-PAGE and immunoblotted using Stat3 mAb. Lane ST represents purified wild type Stat3 (0.6 μg) loaded directly onto the gel as positive control. B and C, mirror resonance affinity assay. Two cells of a biotin-coated cuvette pretreated with saturating amounts of NeutrAvidin. One well of the cuvette was pretreated with biotinylated phosphopeptide based on Tyr-1068 (pY1068, left panel), whereas the other well was pretreated with biotinylated non-phosphorylated peptide Tyr-1068 (Y1068, right panel) as a control for nonspecific binding. Wild type or mutated Stat3 protein was added in the concentrations indicated to each of the two cells, and mirror resonance measurements were recorded continuously for 10 min as shown.View Large Image Figure ViewerDownload (PPT)To test the pocket 1 component of the two models and to ensure that our peptide pull-down system was sufficiently sensitive to detect reduced binding of Stat3 containing mutations in pocket 2, we added either K591L or R609L to the 3M mutant to generate Stat3-3M+K591L and Stat3-3M+R609L. Addition of either mutation resulted in elimination of binding to both Tyr-1068 and Tyr-1086 PDPs indicating that each of the side chains of Lys-591 and Arg-609 make important contributions to binding of the phosphotyrosine.To confirm these findings and to determine whether introduction of the pocket 2 mutations resulted in subtle alterations in kinetics of binding undetectable using phosphopeptide affinity immunoblot analysis, we performed mirror resonance affinity assays using phosphorylated and non-phosphorylated Tyr-1068 dodecapeptide (Fig. 3, B and C, and Table II). Review of the real time mirror resonance affinity curves (Fig. 3, B and C) and kinetic analysis (Table II) revealed undetectable binding of Stat3-3M+Lys-591 and Stat3-3M+Arg-609 to phosphorylated Tyr-1068 dodecapeptide confirming the results of peptide immunoblot analysis. Furthermore, each of the pocket 2 mutant Stat3 proteins examined (3M, 4M, and 6M) demonstrated ka, kd, and KD values for binding to Tyr-1068 PDPs indistinguishable from wild type Stat3, confirming the peptide immunoblot analysis and indicating that Stat3 SH2 binding to the +3 Gln within Tyr-1068 does not require any of the side chains predicted in either of the proposed models.Table IIKinetics of wild type and mutant Stat3 binding to Tyr-1068 PDP determined by mirror resonance biosensor analysisStat3kaaAssociation rate constant determined from slope of line from plot of ka versus [ligand].kdbDissociation rate constant determined from y intercept of plot of ka versus [ligand].KDcDissociation equilibrium constant determined from ratio of kd/ka.m−1 s−1ms−1nmWT30730.72233M33710.61774M2673 ± 481dMean ± S.E. of two separate experiments.0.8 ± 0.3271 ± 486M2619 ± 674dMean ± S.E. of two separate experiments.0.7 ± 0.3249 ± 22a Association rate constant determined from slope of line from plot of ka versus [ligand].b Dissociation rate constant determined from y intercept of plot of ka versus [ligand].c Dissociation equilibrium constant determined from ratio of kd/ka.d Mean ± S.E. of two separate experiments. Open table in a new tab Computational Modeling of Stat3 SH2 Binding to +3 Glu within YXXQ-containing Phosphopeptides—To generate a new and more accurate model for Stat3 SH2 binding to +3 Gln, we used the structure of Tyr-1068 phosphopeptide (EpYINQ), available from its crystal structure bound by Grb2 (28Rahuel J. Garcia-Echeverria C. Furet P. Strauss A. Caravatti G. Fretz H. Schoepfer J. Gay B. J. Mol. Biol. 1998; 279: 1013-1022Google Scholar) (Protein Data Bank code 1ZFP), and the structure of Stat3 from Trp-580 to Leu-670, obtained from the crystal structure of Stat3β bound to DNA (22Becker S. Groner B. Muller C.W. Nature. 1998; 394: 145-151Google Scholar) (Protein Data Bank code 1BG1), to computationally model the interaction with the lowest energy. All energy minimization calculations were carried out under AMBER force field by using the DISCOVER/Insight II program. A total of 300 steps of conjugate gradient energy minimization was performed following rigid hand-docking to fit the Tyr(P) of the EpYINQ peptide into the binding pocket composed of residues Lys-591 and Arg-609 by taking into consideration Van der Waals and Coulomb forces. The interaction between Stat3-SH2 and EpYINQ with the complex lowest energy (Fig. 4A) had a total binding energy of -478.8 kcal/mol. This computational result predicted that the major binding energy for this binding configuration comes from a hydrogen bond interaction involving oxygen within the Tyr(P) +3 Gln side chain and the peptide amide hydrogen at Glu-638 located within a loop region of Stat3 SH2.Fig. 4Revised model of Stat3 SH2 binding to +3 Gln within YXXQ-containing phosphopeptide ligands. A, computational modeling using the Biopolymer program in the Insight II environment was used to perform local energy optimization of the interaction of Stat3 SH2 (shown as a gray ribbon) with phosphopeptide ligand (EpYINQ shown as a green ribbon) based upon the known structures of each. As indicated, the oxygen on the side chain of the Tyr(P) +3 Gln within the EpYINQ peptide is predicted to form a hydrogen (H-bond) bond with the amide hydrogen at Glu-638 and to make a major contribution to the binding energy. The positions are shown for the side chains of Lys-591 and Arg-609 proposed to be major contributors to pocket 1, Glu-638, Tyr-640, and Tyr-657 proposed by Chakraborty to form pocket 2, and for the side chain of Trp-623 proposed to force a β-turn in the peptide ligand. The +3 Gln and Glu-638 are shown as ball-and-stick models, the remaining side chains as stick models; oxygen atoms are shown in red, carbon in gray, nitrogen in blue, and phosphorus in orange. B, overlay of the known structure of wild type Stat3 (green) with the predicted structure of Stat3-E638P (gray). The positions of the side chains of relevant residues are indicated for wild type Stat3 (aqua stick models) and for Stat3E638 (gray stick models).View Large Image Figure ViewerDownload (PPT)To test the contribution of the Glu-638 amide hydrogen, we generated Stat3-E638P by site-directed mutagenesis, which eliminated the amide hydrogen donor predicted to bind with oxygen within the +3 Gln side chain. In consideration of the possible effect of this mutation on secondary structure, we modeled E638P within Stat3-SH2 using Biopolymer in the Insight II environment and carried out local energy minimization as follows: 1) with all residues fixed except for Val-637 to Pro-639 to assess the e
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