The Phosphotyrosine Peptide Binding Specificity of Nck1 and Nck2 Src Homology 2 Domains
2006; Elsevier BV; Volume: 281; Issue: 26 Linguagem: Inglês
10.1074/jbc.m512917200
ISSN1083-351X
AutoresSusanne Frese, Wolf‐Dieter Schubert, A.C. Findeis, Tobias Marquardt, Yvette Roske, Theresia E. B. Stradal, Dirk W. Heinz,
Tópico(s)RNA and protein synthesis mechanisms
ResumoNck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nckα) and Nck2 (Nckβ and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1. Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nckα) and Nck2 (Nckβ and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1. Dynamic processes in eukaryotic cells, such as cellular movement, changes in cell shape, and transport of vesicles, rely on constant remodeling of the actin cytoskeleton. Adapter proteins, essential in transmitting and modulating corresponding stimuli, frequently contain SH2 3The abbreviations used are: SH, Src homology; EPEC, enteropathogenic E. coli; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid; PDGFR, platelet-derived growth factor receptor; r.m.s.d., root mean square deviation; SPR, surface plasmon resonance. domains to recognize and bind tyrosine-phosphorylated motifs. Nck1 (Nckα) and Nck2 (Nckβ or Grb4) are two such adapter proteins (1Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (145) Google Scholar, 2Braverman L.E. Quilliam L.A. J. Biol. Chem. 1999; 274: 5542-5549Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 3Buday L. Wunderlich L. Tamas P. Cell. Signal. 2002; 14: 723-731Crossref PubMed Scopus (193) Google Scholar), both bearing three SH3 domains and a C-terminal SH2 domain (4Lehmann J.M. Riethmuller G. Johnson J.P. Nucleic Acids Res. 1990; 18: 1048Crossref PubMed Scopus (160) Google Scholar). Mice lacking both Nck genes are not viable, underscoring the importance of these adapters (1Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (145) Google Scholar). A high sequence identity (68% overall and 82% for the SH2 domains) and single gene knockouts of Nck1 and Nck2 (1Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (145) Google Scholar) indicate that the function of the proteins may substantially overlap. Both bind receptor tyrosine kinases such as the PDGFR (5Nishimura R. Li W. Kashishian A. Mondino A. Zhou M. Cooper J. Schlessinger J. Mol. Cell. Biol. 1993; 13: 6889-6896Crossref PubMed Scopus (155) Google Scholar) and other tyrosine-phosphorylated proteins via their SH2 domains (3Buday L. Wunderlich L. Tamas P. Cell. Signal. 2002; 14: 723-731Crossref PubMed Scopus (193) Google Scholar). However, Nck1 or Nck2 has also been reported to bind distinct targets. Exclusive Nck2 binders include EphrinB1 (6Cowan C.A. Henkemeyer M. Nature. 2001; 413: 174-179Crossref PubMed Scopus (289) Google Scholar, 7Bong Y.S. Park Y.H. Lee H.S. Mood K. Ishimura A. Daar I.O. Biochem. J. 2004; 377: 499-507Crossref PubMed Scopus (25) Google Scholar), EphrinB2 (8Su Z. Xu P. Ni F. Eur. J. Biochem. 2004; 271: 1725-1736Crossref PubMed Scopus (21) Google Scholar), and Disabled-1 (Dab-1) (9Pramatarova A. Ochalski P.G. Chen K. Gropman A. Myers S. Min K.T. Howell B.W. Mol. Cell. Biol. 2003; 23: 7210-7221Crossref PubMed Scopus (77) Google Scholar), all involved in neuronal signaling. In the case of the PDGFR, Tyr(P)751 is reported to be Nck1-specific (5Nishimura R. Li W. Kashishian A. Mondino A. Zhou M. Cooper J. Schlessinger J. Mol. Cell. Biol. 1993; 13: 6889-6896Crossref PubMed Scopus (155) Google Scholar), whereas Tyr(P)1009 is Nck2-specific (10Chen M. She H. Kim A. Woodley D.T. Li W. Mol. Cell. Biol. 2000; 20: 7867-7880Crossref PubMed Scopus (75) Google Scholar). Furthermore, Nck1 and Nck2 have both been implicated in the infection process of enteropathogenic Escherichia coli (EPEC) (11Gruenheid S. DeVinney R. Bladt F. Goosney D. Gelkop S. Gish G.D. Pawson T. Finlay B.B. Nat. Cell Biol. 2001; 3: 856-859Crossref PubMed Scopus (299) Google Scholar), a frequent cause of severe infant diarrhea (12Chen D.H. Frankel G. FEMS Microbiol. Rev. 2005; 29: 83-98Crossref PubMed Scopus (259) Google Scholar). EPEC adheres tightly to the membrane of intestinal enterocytes inducing massive remodeling of the microfilament system and suppression of microvilli (13Moon H.W. Whipp S.C. Argenzio R.A. Levine M.M. Giannella R.A. Infect. Immun. 1983; 41: 1340-1351Crossref PubMed Google Scholar, 14Clarke S.C. Haigh R.D. Freestone P.P. Williams P.H. Clin. Microbiol. Rev. 2003; 16: 365-378Crossref PubMed Scopus (182) Google Scholar). This involves the "translocated intimin receptor" (Tir), introduced into the host cell by a type III secretion system (11Gruenheid S. DeVinney R. Bladt F. Goosney D. Gelkop S. Gish G.D. Pawson T. Finlay B.B. Nat. Cell Biol. 2001; 3: 856-859Crossref PubMed Scopus (299) Google Scholar). Insertion of Tir into the host cell membrane (15Kenny B. DeVinney R. Stein M. Reinscheid D.J. Frey E.A. Finlay B.B. Cell. 1997; 91: 511-520Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar) provides a binding site to the bacterial outer membrane protein intimin (16Luo Y. Frey E.A. Pfuetzner R.A. Creagh A.L. Knoechel D.G. Haynes C.A. Finlay B.B. Strynadka N.C. Nature. 2000; 405: 1073-1077Crossref PubMed Scopus (250) Google Scholar). Tir clustering induces phosphorylation of its cytosolic C-terminal Tyr474 by an Src family kinase (17Phillips N. Hayward R.D. Koronakis V. Nat. Cell Biol. 2004; 6: 618-625Crossref PubMed Scopus (106) Google Scholar, 18Campellone K.G. Rankin S. Pawson T. Kirschner M.W. Tipper D.J. Leong J.M. J. Cell Biol. 2004; 164: 407-416Crossref PubMed Scopus (104) Google Scholar) and hence recruitment of Nck1 and/or Nck2 through their SH2 domains (11Gruenheid S. DeVinney R. Bladt F. Goosney D. Gelkop S. Gish G.D. Pawson T. Finlay B.B. Nat. Cell Biol. 2001; 3: 856-859Crossref PubMed Scopus (299) Google Scholar). By recruiting and activating N-WASP and hence the Arp2/3 complex through their SH3 domains (11Gruenheid S. DeVinney R. Bladt F. Goosney D. Gelkop S. Gish G.D. Pawson T. Finlay B.B. Nat. Cell Biol. 2001; 3: 856-859Crossref PubMed Scopus (299) Google Scholar, 19Kalman D. Weiner O.D. Goosney D.L. Sedat J.W. Finlay B.B. Abo A. Bishop J.M. Nat. Cell Biol. 1999; 1: 389-391Crossref PubMed Scopus (178) Google Scholar), Nck1 or Nck2 in turn induce actin polymerization producing dynamic bacteria-presenting protrusions of the plasma membrane known as pedestals (20Rosenshine I. Ruschkowski S. Stein M. Reinscheid D.J. Mills S.D. Finlay B.B. EMBO J. 1996; 15: 2613-2624Crossref PubMed Scopus (229) Google Scholar). The vaccinia virus similarly exploits Nck. Following its intracellular replication, the virus is transported to the cell periphery in a microtubule-dependent process (21Rietdorf J. Ploubidou A. Reckmann I. Holmstrom A. Frischknecht F. Zettl M. Zimmermann T. Way M. Nat. Cell Biol. 2001; 3: 992-1000Crossref PubMed Scopus (237) Google Scholar), where the viral envelope protein A36R is tyrosine-phosphorylated at Tyr112 recruiting Nck through its SH2 domain. Again, N-WASP/Arp2/3-induced actin polymerization leads to the formation of motile plasma membrane projections beneath the virus (21Rietdorf J. Ploubidou A. Reckmann I. Holmstrom A. Frischknecht F. Zettl M. Zimmermann T. Way M. Nat. Cell Biol. 2001; 3: 992-1000Crossref PubMed Scopus (237) Google Scholar, 22Lemmon M.A. Ladbury J.E. Biochemistry. 1994; 33: 5070-5076Crossref PubMed Scopus (54) Google Scholar, 23Frischknecht F. Moreau V. Rottger S. Gonfloni S. Reckmann I. Superti-Furga G. Way M. Nature. 1999; 401: 926-929Crossref PubMed Scopus (350) Google Scholar). Here we have investigated the extent to which the SH2 domains of Nck1 and Nck2 differ in phosphotyrosine signaling. By surface plasmon resonance (SPR) spectroscopy, we identify a Tir-derived phosphopeptide as the strongest natural ligand of both Nck1 and Nck2 SH2 domains. By epitope scan, based on the sequential mutation of the Tir sequence, we derive the optimal peptide profile for both SH2 domains. Furthermore, we present the crystal structure of Nck1-SH2 without ligand and that of both Nck1- and Nck2-SH2 in complex with Tir-derived phosphopeptides. Based on our results, we predict new potential interaction partners for the SH2 domains of Nck1 and Nck2 and demonstrate the tyrosine phosphorylation-dependent interaction for one of these, GIT1. Overall, our analyses do not support distinct phosphotyrosine peptide affinities of the SH2 domains to explain distinguishing characteristics of Nck1 and Nck2. Differences must therefore involve regions outside the peptide-binding groove of the SH2 domains. Production of GST Fusion Proteins—The cDNA for Nck and Grb2 proteins was a kind gift from Michael Way (Cancer Research UK, Lincoln's Inn Fields Laboratories, London, UK) and Ottmar Janssen (Institute of Immunology, Kiel, Germany). Coding sequences of the SH2 domains of human Nck1 (amino acids 281-377) and Nck2 (amino acids 284-380) were amplified by PCR and ligated into the vector pGEX-6P-1 (Amersham Biosciences) using primers 5′-GGTCGGATCCCCTTGGTATTATGGCAAAGTCAC-3′ (5′-primer, BamHI restriction site) and 5′-GGCGCTCGAGTCATGATAAATGCTTGACAAGATA-3′ (3′-primer, XhoI restriction site) for Nck1-SH2, and 5′-GGTCGGATCCGAGTGGTACTACGGGAACGTG-3′ (5′-primer, BamHI restriction site) and 5′-GGCGCTCGAGTCACTGCAGGGCCCTGACGAG-3′ (3′-primer, XhoI restriction site) for Nck2-SH2. GST fusions of Nck and Grb2 SH2 domains were produced in E. coli strain BL21-CodonPlus (Stratagene) at 22 °C overnight. Cells were centrifuged, resuspended in 50 ml of PBS, and disrupted by a French press. The supernatants were incubated with glutathione-Sepharose (Amersham Biosciences) and washed with PBS. For pulldown assays, PBS plus 10% glycerol allowed snap-freezing in liquid nitrogen. For all other assays, the GST/Nck-SH2 fusion protein was eluted using 10 mm glutathione in PBS (Nck1) or 20 mm Tris/HCl, pH 8.0, 200 mm NaCl (Nck2) and either used as such in peptide overlay studies or cleaved using PreScission protease (Amersham Biosciences). Further purification included Mono Q anion exchange (20 mm citrate buffer, pH 6.0, Nck1) and gel filtration chromatography (20 mm citrate buffer, pH 6, 200 mm NaCl for Nck1 and 20 mm Tris/HCl, 200 mm NaCl for Nck2). Crystallization and Data Collection—Nck1-SH2 was crystallized by hanging drop vapor diffusion at 4 °C by using 6-10 mg/ml protein in 20 mm Tris/HCl, pH 8.0, and 25% PEG5000 MME, 0.1 m MES, pH 6.5, 0.2 m (NH4)2SO4, 0.1 m guanidine HCl as precipitant. 20% MPD in mother liquor served as cryoprotectant. For co-crystallization, protein and peptide were mixed in a ratio of 1:1.1. Nck1-SH2:Tir12, where Tir12 is a 12-residue, chemically synthesized peptide derived from the EPEC-protein Tir. The complex was crystallized in 2.4 m (NH4)2HPO4, 0.1 m Tris, pH 8.5; Nck2-SH2/Tir8 in 50% MPD, 15% ethanol, and 0.01 m Na acetate. Diffraction data were collected at 100K at beamlines BL2 (Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung m.b. H, Berlin, Germany) and BW6 (Max-Planck-Gesellschaft, Deutsches Elektronen-Synchrotron, Hamburg, Germany). Data collection statistics are summarized in Table 1.TABLE 1Binding affinities of Nck1- and Nck2-SH2 for phosphorylated peptides Binding constants for peptides from bacterial (Tir, A36R) and human (EphrinB1/2, PDGFR, and Dab-1) interaction partners, reported to recruit either Nck1- or Nck2-SH2, were determined by SPR. The phosphotyrosine sequence number serves to distinguish peptides from the same protein.Peptide sequenceBinding constantNck1-SH2Nck2-SH2μmTir10EHIpYDEVAAD0.06 (±0.01)0.37 (±0.04)Tir10 (unphosphorylated)EHIYDEVAADNo bindingNo bindingPDGFR751ESVDpYVPMLDMKNo bindingNo bindingPDGFR1009SSVLpYTAVQPNE27 (±1)230 (±60)EphrinB1/2CPHpYEKVSGD22 (±3)130 (±50)Dab-1 (220)ENIpYQVPTSQNo bindingNo bindingDab-1 (232)EGVpYDVPKSQNo bindingNo bindingA36REHIpYDSVAGS59 (±9)150 (± 70) Open table in a new tab Structure Determination—Data were processed using the HKL (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar) and CCP4 (25Collaborative Gomputational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) suites. Structures were solved by molecular replacement using EPMR (26Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (691) Google Scholar) and Grb2 as a search model for Nck1-SH2 and the latter for both protein-peptide complexes. The structures were refined by CNS using rigid-body and simulated annealing protocols (27Bruenger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) and REFMAC5 (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar). Matching subsets of diffraction data (5%) were set aside for Rfree calculation (29Bruenger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3872) Google Scholar). The programs O (30Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 2: 110-119Crossref Scopus (13014) Google Scholar) and Coot (31Emsley P. Cowtan K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar) were used for manual model building and structural analysis. Structures were validated using PROCHECK (32Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and WHATIF (33Vriend G. J. Mol. Graphics. 1990; 8 (52-56): 29Google Scholar). Figs. 1 and 2 were prepared using MOLSCRIPT (34Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), GRASP (35Nicholls A. Sharp K. Honig B. Proteins Struct. Funct. Genet. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar), and POV-Ray and Fig. 3 using PYMOL.FIGURE 2Phosphopeptide complexes of Nck1-SH2 and Nck2-SH2. A, Nck1-SH2 binds Tir12 (yellow), a 12-residue peptide from the EPEC virulence factor Tir. Two N-terminal glutamate residues of the peptide, not resolved in the electron density, are excluded. The electrostatic surface reveals a deep, positively charged (blue) phosphotyrosine binding pocket. B, Nck2-SH2 complexed to Tir8 (green). C, schematic representation of the Nck-SH2/Tir interaction. Chemical bonds of Tir12 are shown in black, and those of residues of Nck are shown in orange. Hydrogen bonds are indicated by dashed lines, hydrophobic interactions by green arcs, and a cation-π interaction by a blue dashed line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Nck1-SH2-Tir12 complex (shades of yellow) superimposed on Nck2-SH2/Tir8 (green). A, the interactions of SH2 domain and peptide are conserved superimposing the peptides. B, the 17 nonconserved residues between Nck1- and Nck2-SH2 (green spheres) are distributed over the SH2 surfaces away from the peptide-protein interface.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Epitope Scanning—233 Tir-derived dodecapeptides (11 positions × 20 amino acids, 11 negative control peptides with unphosphorylated Tyr, plus 1 positive and 1 negative control peptide; see supplemental material) were generated by spot synthesis on cellulose as solid support. The membrane was blocked by overnight incubation with 10 ml of MBS (2 ml of blocking reagent (Sigma-Genosys), 0.5 g of saccharose, pH 7, and 8 ml of T-TBS (8.0 g/liter NaCl, 0.2 g/liter KCl, 6.1 g/liter Tris base, 0.05% Tween, pH 8.0)). GST-Nck1- or GST-Nck2-SH2 was incubated with the peptide membrane (5 μg/ml in MBS) for 2 h. After a brief T-TBS rinse, the membrane was incubated with a horseradish peroxidase-conjugated GST antibody for 1 h. After two washing steps, bound antibody was detected via development with the ECL kit (Roche Applied Science). The experiment was performed in triplicate. Intensity of spots was quantified by luminometry employing a CCD camera (Fuji) and analyzed using AIDA software (Raytest, Germany). Spot intensities of each experiment were normalized by division through the means of positive control intensities (original Tir12-peptides). Normalized intensities were set to percentages (mean of Tir12-peptides: 100%) and averaged over all three experiments. Surface Plasmon Resonance—Surface plasmon resonance experiments were performed using a BIAcore 2000 (BIAcore AB, Uppsala, Sweden) at 25 °C, and a flow rate of 10 μl/min. Biotinylated decapeptides were passed over and allowed to bind to the surface of a streptavidin-coated sensor chip (SA5, Pharmacia Biosensor). A dilution series of ∼1 mm to 10 nm Nck1- and Nck2-SH2 was prepared in running buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 3 mm EDTA, and 0.005% Tween 20). Protein association and dissociation were each recorded for 2 min for each concentration. The surface was regenerated between injections using 1 m NaCl. All experiments were performed in triplicate. Sensorgrams for each protein concentration were aligned to the same base line after subtraction of the reference. The sensorgram data were quantified by plotting the resonance units at equilibrium (Req) against the protein concentration. The binding constant KD was determined by curve fitting to a 1:1 binding isotherm as shown in Equation 1, Req[Nck]=Rmax·[Nck]KD+[Nck](Eq. 1) where Req indicates the resonance units at equilibrium; KD is the dissociation constant; [Nck] is the molar concentration of Nck, and Rmax is the maximal resonance signal. Pulldown Assays—A431 cells (ATCC, CRL 1555) and HeLa S3 cells (CCl-2.2) were maintained as recommended by the supplier (ATCC, LGC Promochem, Wesel, Germany). 10-cm diameter dishes of 70% confluent cells were serum-deprived for 24 h and treated with Dulbecco's modified Eagle's medium in the presence or absence of 2 μm phorbol 12-myristate 13-acetate for 10 min at 37 °C to stimulate protein phosphorylation. Cells were lysed in 500 μl of ice-cold lysis buffer (12 mm Tris, 16 mm HEPES, pH 7.4, 50 mm NaCl, 15 mm KCl, 20 mm NaF, 1 mm NaVO4) for 10 min on ice and harvested with a cell scraper. The lysate was cleared at 15,000 × g for 15 min at 4 °C and incubated for 1 h at 4 °C on a rotary wheel with 25 μl of glutathione-Sepharose loaded with GST or GST-SH2 fusions of Grb2, Nck1, or Nck2. Precipitates were washed twice, resolved by SDS-PAGE, and analyzed by immunoblotting. Anti-GIT1 was purchased from Santa Cruz Biotechnology. Binding Affinities of Nck1- and Nck2-SH2 for Target-derived Phosphopeptides—We have determined the binding affinities of the sequentially related SH2 domains of Nck1 and Nck2 (Fig. 1A) for reported binding partner peptides by surface plasmon resonance spectroscopy using chemically synthesized phosphopeptides, purified by high pressure liquid chromatography and verified by mass spectrometry. Peptides include those of the exogenous virulence factors Tir (EPEC) and A36R (vaccinia) and the endogenous interaction partners PDGFR, Dab-1, EphrinB1, and EphrinB2. Dab-1 and PDGFR each contain two phosphorylation sites, Tyr220 and Tyr232 for Dab-1, Tyr751 and Tyr1009 for PDGFR, and one for both EphrinB1 and -B2 (Tyr298 and Tyr304). Dissociation constants (KD) for various peptides were determined by analyzing the association and dissociation of Nck1- or Nck2-SH2 at various concentrations to peptide-coated chips (Table 1). With a KD of 60 nm and 370 nm for Nck1 and Nck2, both SH2 domains bind the Tir-derived phosphopeptide with high affinity. This is similar to the 313 nm observed for Nck1-SH2/Tir-phosphopeptide determined in an earlier study using fluorescence polarization (11Gruenheid S. DeVinney R. Bladt F. Goosney D. Gelkop S. Gish G.D. Pawson T. Finlay B.B. Nat. Cell Biol. 2001; 3: 856-859Crossref PubMed Scopus (299) Google Scholar). As expected, neither SH2 domain binds to the nonphosphorylated Tir peptide, a negative control. Binding to the A36R-derived peptide is ∼103-fold weaker (KD of 59 μm, 150 μm for Nck1/Nck2) than to the Tir-derived peptide but similar to those for PDGFR Tyr(P)1009 (27 μm/230 μm) and EphrinB1/2 (22 μm/130 μm) peptides. Unexpectedly, binding of Nck1- or Nck2-SH2 to Dab-1 Tyr(P)220 and Tyr(P)232 and PDGFR Tyr(P)751 peptides was not observed, contradicting their identification as Nck1 and Nck2 SH2 ligands. Overall, the isolated Nck2-SH2 displays a slightly weaker affinity (2-9-fold) than Nck1-SH2. Nevertheless, both proteins bind to the same peptides, and the trend in binding affinities is similar. Crystal Structures, Ligand-free SH2 Domain of Nck1—We started to crystallize both domains alone and in complex with tightly binding Tir-derived phosphopeptide to identify potential differences in phosphopeptide binding of Nck1 and Nck2 SH2 domains. The ligand-free structure of Nck1-SH2 at 1.8 Å resolution (Fig. 1B and Table 2) (Protein Data Bank code 2CI8) is broadly similar to that of other SH2 domains (36Bradshaw J.M. Waksman G. Adv. Protein Chem. 2002; 61: 161-210Crossref PubMed Scopus (103) Google Scholar). It consists of a central β-sheet (βB, βC, and βD) flanked by two α-helices (αA and αB). Compared with other SH2 domains, Nck1-SH2 has a second β-sheet (βF′/βF″) near the domain surface, also observed in the recent NMR structure of Nck2-SH2 (37Ran X. Song J. J. Biol. Chem. 2005; 280: 19205-19212Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In our crystal, we find that Nck1-SH2 forms a domain-swapped dimer. The loop D′E that connects β-strands D′ and E adopts an extended conformation separating N- and C-terminal halves of the domain and allowing two domains to form a symmetrical dimer. The domain swapped dimeric form of Nck1-SH2 was not detected in solution by gel filtration, dynamic light scattering, and analytical ultracentrifugation, 4S. Frese, W.-D. Schubert, A. C. Findeis, T. Marquardt, Y. S. Roske, T. E. B. Stradal, and D. W. Heinz, unpublished data. indicating that this was probably caused by 0.1 m guanidine hydrochloride in the crystallization solution, and thus is not biologically relevant (also see supplemental material).TABLE 2Data collection and refinement statisticsData setNck1-SH2Nck1-SH2/Tir12Nck2-SH2/Tir8 Space groupC222P212121P212121 Unit cell (a, b, and c in Å)62.9, 82.5, 44.555.1, 60.5, 65.131.3, 58.2, 52.3 Monomers/asymmetric unit12 + 21 + 1 Wavelength (Å)0.921.051.05 Resolution rangeaValues in parentheses indicate shell of highest resolution50–1.80 (1.83–1.80)30–1.40 (1.42–1.40)50–1.45 (1.48–1.45) RmergeaValues in parentheses indicate shell of highest resolution (%)5 (37)4 (31)4 (31) I/σ(I)aValues in parentheses indicate shell of highest resolution34.3 (3.2)28.1 (4.2)25.1 (3.7) CompletenessaValues in parentheses indicate shell of highest resolution (%)99.1 (95.5)96.4 (88.1)98.4 (96.7) RedundancyaValues in parentheses indicate shell of highest resolution6.1 (4.6)4.0 (3.7)3.3 (2.9) Unique reflectionsaValues in parentheses indicate shell of highest resolution11,119 (490)38,419 (2052)17,053 (818)Refinement Resolution rangeaValues in parentheses indicate shell of highest resolution (Å)30–1.80 (1.85–1.80)28–1.50 (1.54–1.50)20–1.45 (1.49–1.45) R (%)aValues in parentheses indicate shell of highest resolution22 (23)20 (23)15 (20) Rfree (%)aValues in parentheses indicate shell of highest resolution24 (26)24 (29)18 (25) No. of reflectionsaValues in parentheses indicate shell of highest resolution10,457 (753)28,813 (2419)16,143 (1070) No. of water molecules65246117 Average B-factor (Å2)261115 r.m.s.d. bond lengths (Å)0.0330.0210.025 r.m.s.d. bond angles (°)2.62.01.9 Ramachandran plot (%)bFavored/generous/additional/disallowed regions92/8/0/091/8/1/095/5/0/0a Values in parentheses indicate shell of highest resolutionb Favored/generous/additional/disallowed regions Open table in a new tab The Mode of Ligand Binding Is Conserved between Nck1 and Nck2 SH2 Domains—Nck1- and Nck2-SH2 were co-crystallized with Tir-derived phosphopeptides of different length (Table 2), 12 residues (Tir12, EEHIY*DEVAADP) for Nck1 (Fig. 2A) (Protein Data Bank code 2CI9) and 8 residues (Tir8, HIY*DEVAA) for Nck2 (Fig. 2B) (Protein Data Bank code 2CIA). Peptide binding does not significantly affect the conformation of the SH2 domains. A relatively high root mean square deviation (r.m.s.d.) of 0.9 Å for C-α atoms between ligand-free Nck1-SH2 and Tir12-bound Nck1-SH2 is primarily caused by adjustments in the C terminus as well as flexible loops βB-βC, βC-βD, and βF′-βF″ not involved in peptide binding. Similarly, the SH2 domains of peptide-bound Nck1- and Nck2-SH2 are again highly similar despite an r.m.s.d. of 0.7 Å caused by deviations in the loops described above (Fig. 3). Residues involved in phosphopeptide binding are strictly conserved. As a result, interactions between the peptides and the Nck peptide-binding site are mostly equivalent ((Figs. 2C and 3A) corroborating the finding that both SH2 domains prefer the same substrates. Nonconserved residues are confined to loops (Fig. 3B) not involved in phosphopeptide binding. As observed previously for other SH2 domains (36Bradshaw J.M. Waksman G. Adv. Protein Chem. 2002; 61: 161-210Crossref PubMed Scopus (103) Google Scholar), the phosphate group of Tyr(P) is tightly bound in a hydrophilic pocket (Fig. 2). Two conserved arginines (Arg-αA2, Arg-βB5), three serines (Ser-βB7, Ser-βC2, and Ser-βC3) and the amide N of Glu-βC1 ensure tight coordination (Fig. 2C). Apart from the phosphate group, Tyr(P) is also held in position by hydrophobic interactions of the aromatic ring with Lys-βD6 and Arg-αA2 (cation-π interaction and stacking). Other phosphopeptide residues involved in binding include Val+3 (three residues C-terminal of Tyr(P)), Ala+4, Ile-1, and His-2, recognized by specific interactions including salt bridges, hydrogen bonds, and van der Waals interactions. Specific interactions for residues outside the eight central amino acids include a salt bridge of Arg-βF1 to Asp+6 as seen in the complex of Nck1-SH2 with Tir12. Phosphopeptide Residue Profiles Are Virtually Identical for Nck1-SH2 and Nck2-SH2—To establish the contribution of each phosphopeptide residue to the interaction with Nck SH2 domains, we systematically replaced each residue of the peptide (except Tyr(P)) by all 20 standard amino acids (cysteine was chemically protected by an acetamidomethyl group). An epitope scan method was employed to determine the relative binding affinities of Nck1 and Nck2 SH2 domains for these 201 12-residue phosphopeptides. GST-SH2 fusion proteins were allowed to bind to peptides immobilized to a cellulose matrix, and binding affinity was inferred by the signal intensity of each spot after chemiluminescent detection (supplemental Fig. 2A). The experiment was conducted in triplicate, and values for each peptide were averaged following normalization according to the signal derived from the original Tir peptide (red letters or boxes in supplemental Fig. 2, A and B). The peptide scans for Nck1- and Nck2-SH2 are summarized in Fig. 4, A and B. For each position of the phosphopeptide,
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