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• W2027663241 abstract "Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.Background: Src homology 2 (SH2) domains are known to specifically bind to phosphotyrosine followed by a few amino acids.Results: A novel interaction region was revealed by the solution structure of the C-terminal Src kinase SH2 domain in complex with the Csk-binding protein.Conclusion: The novel interaction region was required for tumor suppression.Significance: The structure sheds new light on the interaction mode of SH2 domains. Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. Background: Src homology 2 (SH2) domains are known to specifically bind to phosphotyrosine followed by a few amino acids. Results: A novel interaction region was revealed by the solution structure of the C-terminal Src kinase SH2 domain in complex with the Csk-binding protein. Conclusion: The novel interaction region was required for tumor suppression. Significance: The structure sheds new light on the interaction mode of SH2 domains. Src homology 2 (SH2) 3The abbreviations used are: SH2, Src homology 2; Csk, C-terminal Src kinase; Cbp, Csk-binding protein; PAG, phosphoprotein associated with glycosphingolipid; HSQC, heteronuclear single quantum correlation; PDB, Protein Data Bank; r.m.s.d., root mean square deviation; SFK, Src family kinase. domains are noncatalytic regions commonly observed in various types of signal transduction proteins. They function as modules that mediate the interaction between proteins by recognizing a phosphotyrosine (Tyr(P)) in the target proteins. Structural and quantitative binding analyses of many SH2 domains in complexes with related ligand peptides have shown that SH2 domains generally recognize Tyr(P) with three amino acid residues toward the C terminus (Tyr(P)-Xaa1-Xaa2-Xaa3) of the target ligands using two recognition pockets on the surface of the SH2 domains (1Waksman 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, 2Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. SH2 domains recognize specific phosphopeptide sequences.Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2381) Google Scholar, 3Schlessinger J. Lemmon M.A. SH2 and PTB domains in tyrosine kinase signaling.Sci. STKE 2003. 2003; : RE12Google Scholar, 4Pawson T. Nash P. Assembly of cell regulatory systems through protein interaction domains.Science. 2003; 300: 445-452Crossref PubMed Scopus (1151) Google Scholar). One pocket recognizes Tyr(P) of the target primarily through electrostatic interactions and hydrogen bonds, whereas the other recognizes the remaining three amino acids (Xaa1-Xaa2-Xaa3) specifically through hydrophobic interactions. This specificity is considered to generate versatility in the interaction of SH2 domains (5Piccione E. Case R.D. Domchek S.M. Hu P. Chaudhuri M. Backer J.M. Schlessinger J. Shoelson S.E. Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding.Biochemistry. 1993; 32: 3197-3202Crossref PubMed Scopus (134) Google Scholar, 6Ladbury J.E. Arold S. Searching for specificity in SH domains.Chem. Biol. 2000; 7: R3-R8Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). However, it is still controversial whether the latter pocket alone is sufficient to determine the specificity of the associated interactions (4Pawson T. Nash P. Assembly of cell regulatory systems through protein interaction domains.Science. 2003; 300: 445-452Crossref PubMed Scopus (1151) Google Scholar, 7Bradshaw J.M. Mitaxov V. Waksman G. Mutational investigation of the specificity determining region of the Src SH2 domain.J. Mol. Biol. 2000; 299: 521-535Crossref PubMed Scopus (25) Google Scholar). The protein C-terminal Src kinase (Csk) includes SH3, SH2, and kinase domains. This kinase specifically phosphorylates a regulatory Tyr in the C-terminal tail of Src Tyr kinases (SFKs) (8Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Constitutive activation of Src family kinases in mouse embryos that lack Csk.Cell. 1993; 73: 1125-1135Abstract Full Text PDF PubMed Scopus (363) Google Scholar, 9Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src.Nature. 1991; 351: 69-72Crossref PubMed Scopus (510) Google Scholar). This event leads to an intramolecular interaction between the Tyr(P)-containing tail and the SH2 domain of the phosphorylated SFK, shifting it to an inactive closed conformation. Thus, Csk negatively regulates the kinase activity of SFKs and plays an important role in physiological functions via signaling pathways for cell proliferation, differentiation, adhesion, and migration (10Chow L.M. Fournel M. Davidson D. Veillette A. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk.Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar). Although SFKs are anchored to membranes via their fatty-acylated N termini, Csk, which lacks such a fatty acylation site, exists in the cytoplasm (9Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src.Nature. 1991; 351: 69-72Crossref PubMed Scopus (510) Google Scholar). Thus, for Csk to efficiently access SFKs, Csk interacts with the Csk-binding protein (Cbp/PAG), which is localized to membrane microdomains enriched in cholesterol, glycosphingolipids, and lipid rafts, and it is subsequently recruited to the reaction space (11Kawabuchi M. Satomi Y. Takao T. Shimonishi Y. Nada S. Nagai K. Tarakhovsky A. Okada M. Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases.Nature. 2000; 404: 999-1003Crossref PubMed Scopus (463) Google Scholar, 12Brdicka T. Pavlistová D. Leo A. Bruyns E. Korínek V. Angelisová P. Scherer J. Shevchenko A. Hilgert I. Cerný J. Drbal K. Kuramitsu Y. Kornacker B. Horejsí V. Schraven B. Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein-tyrosine kinase csk and is involved in regulation of T cell activation.J. Exp. Med. 2000; 191: 1591-1604Crossref PubMed Scopus (405) Google Scholar). This interaction is known to occur between the SH2 domain of Csk and the SFK-phosphorylated Tyr-314 of Cbp, but it remains unknown whether any other region in Cbp is involved in the interaction (13Takeuchi S. Takayama Y. Ogawa A. Tamura K. Okada M. Transmembrane phosphoprotein Cbp positively regulates the activity of the carboxyl-terminal Src kinase, Csk.J. Biol. Chem. 2000; 275: 29183-29186Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In addition, unlike SFKs, which conserve specific Tyr in their C-terminal tails for regulating activity, Csk lacks such a functional tail (9Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src.Nature. 1991; 351: 69-72Crossref PubMed Scopus (510) Google Scholar). Nevertheless, the crystal structure of Csk by itself exhibited the existence of two conformers corresponding to active and inactive forms (14Ogawa A. Takayama Y. Sakai H. Chong K.T. Takeuchi S. Nakagawa A. Nada S. Okada M. Tsukihara T. Structure of the carboxyl-terminal Src kinase, Csk.J. Biol. Chem. 2002; 277: 14351-14354Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The mechanism of Csk activation is described in detail in a previous review (15Ia K.K. Mills R.D. Hossain M.I. Chan K.C. Jarasrassamee B. Jorissen R.N. Cheng H.C. Structural elements and allosteric mechanisms governing regulation and catalysis of CSK-family kinases and their inhibition of Src-family kinases.Growth Factors. 2010; 28: 329-350Crossref PubMed Scopus (21) Google Scholar). The SH2 domain appears to be required for stabilizing the active form of Csk through connection between the βB/βC loop (SH2) and the β3/αC loop (kinase). It was also reported that Csk activity is increased through interactions with phosphorylated Cbp or its peptides (13Takeuchi S. Takayama Y. Ogawa A. Tamura K. Okada M. Transmembrane phosphoprotein Cbp positively regulates the activity of the carboxyl-terminal Src kinase, Csk.J. Biol. Chem. 2000; 275: 29183-29186Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 16Wong L. Lieser S.A. Miyashita O. Miller M. Tasken K. Onuchic J.N. Adams J.A. Woods Jr., V.L. Jennings P.A. Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.J. Mol. Biol. 2005; 351: 131-143Crossref PubMed Scopus (57) Google Scholar, 17Lin X. Ayrapetov M.K. Lee S. Parang K. Sun G. Probing the communication between the regulatory and catalytic domains of a protein-tyrosine kinase, Csk.Biochemistry. 2005; 44: 1561-1567Crossref PubMed Scopus (19) Google Scholar, 18Mikkola E.T. Gahmberg C.G. Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.J. Mol. Biol. 2010; 399: 618-627Crossref PubMed Scopus (12) Google Scholar, 19Lin X. Wang Y. Ahmadibeni Y. Parang K. Sun G. Structural basis for domain-domain communication in a protein-tyrosine kinase, the C-terminal Src kinase.J. Mol. Biol. 2006; 357: 1263-1273Crossref PubMed Scopus (23) Google Scholar). Therefore, it can be speculated that Cbp binding shifts the dynamic equilibrium of at least these two conformers to the more active form. However, the mechanism through which Cbp binding increases Csk activity remains unclear. To elucidate these mechanisms, we first analyzed the interaction of Csk with various lengths of Tyr-314-containing regions of Cbp using gel filtration chromatography. On the basis of this biochemical result, we determined the tertiary solution structure of the complex of Csk-SH2 with a region of Cbp that contained both Tyr-296 and Tyr(P)-314 using liquid-state nuclear magnetic resonance (NMR) spectroscopy. We found that Csk recognizes not only the four canonical amino acids beginning with Tyr(P)-314 but also a region on the N-terminal side of Tyr(P)-314 that contains Tyr-296. The DNA fragment for rat Cbp peptide (from 289 to 321; Cbp5) was amplified using the pGEX-6P-1 plasmid (GE Healthcare) containing the DNA fragment encoding the region from 195 to 328 of Cbp (Cbp3) by PCR using 5′-CGC GGA TCC AAG AGA TTT AGT TCC TTG TCA-3′ and 5′-GGC GAA TTC CTA TCC AGG CTT ATT CAC TGA AGA-3′ as primers (Fig. 1A). The amplified DNA fragment was cleaved with BamHI and EcoRI, and the resulting gene was inserted into the BamHI-EcoRI site of pGEX-6P-1. The pGEX-6P-1 plasmid containing the DNA fragment encoding the region from 302 to 321 of Cbp (Cbp6) or a mutant of Cbp5 (Y296F) was produced by the same method as described above using the following primers: 5′-CGC GGA TCC CCA ACT CTT ACA GAA GAG GAG-3′ and 5′-GGC GAA TTC CTA TCC AGG CTT ATT CAC TGA AGA-3′ for Cbp6 and 5′-CGC GGA TCC AAG AGA TTT AGT TCC TTG TCA TTC AAG TCT CGA-3′ and 5′-GGC GAA TTC CTA TCC AGG CTT ATT CAC TGA AGA-3′ for Cbp5-Y296F. The DNA fragment encoding the region from 312 to 321 of Cbp (Cbp7) was amplified by annealing 5′-GATCC ATG TAT TCT TCA GTG AAT AAG CCT GGA TAG G-3′ and 5′-ATTTC CTA TCC AGG CTT ATT CAC TGA AGA ATA CAT G-3′, and the amplified DNA fragment was inserted into the BamHI-EcoRI site of pGEX-6P-1. For a doubly phosphorylated Tyr(P)-296/Tyr(P)-314 peptide, a synthetic product (TORAY) was used. Escherichia coli BL21(DE3) cells were transformed with pGEX-6P-1 containing DNA encoding each of the Cbp peptides and grown in LB media. The cells were incubated at 25 °C with shaking at 240 rpm for 12 h. The expression of GST-fused Cbp peptides was induced by addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 0.1 mm when the absorbance at 600 nm was between 0.3 and 0.6. The cells were further incubated overnight, harvested by centrifugation, and stored at −80 °C. For purification, the cells were dissolved at 4 °C and disrupted by sonication in 100 mm Tris-HCl buffer (pH 7.4) containing 150 mm NaCl, 1 mm EDTA, 5 mm β-mercaptoethanol, and 1% Nonidet P-40 (lysis buffer). After centrifugation, the supernatant was applied to the GSTrap FF affinity column (GE Healthcare), and adsorbed proteins were eluted using lysis buffer (pH 9.0) containing 20 mm reduced glutathione. All purified GST-Cbp fusion proteins were phosphorylated using recombinant Fyn (Millipore) in 50 mm Tris-HCl buffer (pH 7.4) containing 3 mm MgCl2, 1 mm β-mercaptoethanol, and 4 mm ATP. Full-length (amino acids 1–450) rat Csk was expressed using a baculovirus vector in Sf9 insect cells, as described previously (13Takeuchi S. Takayama Y. Ogawa A. Tamura K. Okada M. Transmembrane phosphoprotein Cbp positively regulates the activity of the carboxyl-terminal Src kinase, Csk.J. Biol. Chem. 2000; 275: 29183-29186Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Cells were lysed using the above-mentioned lysis buffer containing EDTA-free protease inhibitor mixture (Nacalai Tesque) and disrupted using a Dounce homogenizer. The supernatant was collected by centrifugation and applied to the HiTrap Q HP anion exchange column (GE Healthcare) equilibrated with 50 mm Tris-HCl buffer (pH 8.0) containing 1 mm EDTA, 5% glycerol, 5 mm β-mercaptoethanol, and 0.02% octyl-d-glucoside (buffer A). The protein was eluted with a linear gradient of 75–300 mm NaCl. Protein-containing fractions were applied to the HiTrap SP HP cation exchange column (GE Healthcare) equilibrated with buffer A. The protein, eluted with a linear gradient of 50–300 mm NaCl, was applied to the Superdex 200 gel filtration column (GE Healthcare). To obtain 13C- and 15N-labeled proteins, E. coli Origami B(DE3) cells were transformed with pGEX-6P-1(GE Healthcare) containing the gene of Csk-SH2 and were grown in M9 minimal medium containing 1.5 g/liter 15NH4Cl and 2.0 g/liter d-[U-13C6]glucose, as nitrogen and carbon sources, respectively. For the expression of uniformly 15N-labeled proteins, d-[U-13C6]glucose was replaced with 4.0 g/liter d-glucose, and 0.1% glycerol was added to the minimal medium. Cells were incubated at 37 °C with shaking. The expression of proteins was induced by the addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 0.5 mm when the absorbance of cells reached an A600 of 0.6. Bacteria were grown for an additional 3 h at 37 °C. Cells were collected by centrifugation and disrupted by sonication in 50 mm Tris-HCl buffer (pH 7.0) containing 400 mm NaCl, 0.1% Tween 20, 1 mm EDTA, and 1 ml of protease inhibitor mixture (Sigma). After centrifugation, the supernatant solution was passed through the DEAE-Sepharose anion exchange column (GE Healthcare) to remove contaminating nucleic acids. The eluate was applied to the GSTrap FF column (GE Healthcare), and the proteins were eluted with 50 mm Tris-HCl buffer (pH 8.0) containing 10 mm reduced glutathione. The eluate was dialyzed for a few hours at 4 °C against 2 liters of the sonication buffer. After addition of 20 μl of PreScission protease (2 units/μl; GE Healthcare) to the solution, it was further dialyzed over 17 h for cleavage of the fusion proteins. In some cases, GST was cut off by the same protease before elution of the fusion protein from the GSTrap FF column during overnight incubation at 4 °C. The proteins were concentrated using an Amicon ultra-4-centrifugal filter unit (Millipore; molecular cutoff, 3000) and were applied to the Superdex 75 gel filtration column (GE Healthcare) with a 20 mm sodium phosphate buffer solution (pH 6.0) containing 50 mm NaCl. The obtained protein (Csk-SH2) ranged from Met-80 to Met-173 with a tag derived from the expression vector (H2N-Gly-Pro-Leu-Gly-Ser-) attached to the N terminus. Protein concentration was estimated using absorbance at 280 nm (A280) with the calculated molar absorption coefficient of 16,000. The Cbp5 peptide was expressed in E. coli BL21(DE3) cells (Takara) as a fusion protein with GST, which was labeled with 13C- and 15N-stable isotopes. The procedures for expression and purification were almost the same as those for Csk-SH2. After elution from the GSTrap FF column, the eluate was diluted with the same amount of 20 mm Tris-HCl buffer (pH 8.0). The solution was applied to the HiTrap DEAE FF anion exchange column (GE Healthcare), and the Cbp peptide was phosphorylated by addition of Fyn and ATP prior to removal of GST. A solution containing Csk-SH2 and Cbp5 was applied to the gel filtration column, and the fractions containing the complex were collected. Each peptide was mixed with equimolar intact Csk or Csk-SH2 in 100 mm Tris-HCl buffer (pH 8.5) containing 150 mm NaCl or 1 m (NH4)2SO4, 5% glycerol, 5 mm β-mercaptoethanol, and 0.02% octyl-d-glucoside with or without ATP. The solution was applied to the Superdex 200 HR 10/30 (GE Healthcare) gel filtration column; A280 was monitored, and each fraction was analyzed by SDS-PAGE. The synthesized peptide and Csk were mixed in a molar ratio of 2:1, and the mixture was assayed as described above. All NMR spectra were acquired at 298 K, except the three-dimensional aromatic 13C-edited nuclear Overhauser effect spectroscopy (NOESY), which was performed at 288 K (20Lin Z. Xu Y. Yang S. Yang D. Sequence-specific assignment of aromatic resonances of uniformly 13C,15N-labeled proteins by using 13C- and 15N-edited NOESY spectra.Angew. Chem. Int. Ed. Engl. 2006; 45: 1960-1963Crossref PubMed Scopus (28) Google Scholar), using Bruker DRX-500 and DRX-600 instruments equipped with shielded triple-axis gradient triple-resonance probes, and DRX-800, AvanceII-800, and AvanceIII-950 instruments equipped with z axis gradient triple resonance cryogenic probes. For assignments of 1H, 13C, and 15N resonances, a series of two- and three-dimensional experiments were performed (21Bax A. Multidimensional nuclear-magnetic-resonance methods for protein studies.Curr. Opin. Struct. Biol. 1994; 4: 738-744Crossref Scopus (192) Google Scholar). Two-dimensional 1H-15N heteronuclear single- quantum correlation (HSQC), three-dimensional HNCACB, CBCA(CO)NH, HNCA, HN(CO)CA, HNCO, HN(CA)CO, and HBHA(CBCACO)NH spectra were acquired for assignment of backbone signals. For assignment of the aliphatic side-chain signals, two-dimensional 1H-13C constant time HSQC, three-dimensional 15N-edited total correlation spectroscopy (TOCSY) with a mixing time of 79.4 ms, HCCH-TOCSY with a mixing time of 22.6 ms, and C(CO)NH and H(CCO)NH with a mixing time of 22.6 ms spectra were used. For assignment of the aromatic side-chain signals, two-dimensional 1H-1H double-quantum filtered correlation spectroscopy (22Piantini U. Sorensen O.W. Ernst R.R. Multiple quantum filters for elucidating NMR coupling networks.J. Am. Chem. Soc. 1982; 104: 6800-6801Crossref Scopus (1887) Google Scholar, 23Shaka A.J. Freeman R. Simplification of NMR-spectra by filtration through multiple-quantum coherence.J. Magn. Reson. 1983; 51: 169-173Google Scholar, 24Derome A.E. Williamson M.P. Rapid-pulsing artifacts in double-quantum-filtered cosy.J. Magn. Reson. 1990; 88: 177-185Google Scholar) and three-dimensional aromatic 13C-edited NOESY with a mixing time of 100 ms were used. The chemical shifts of the 1Hδ/ϵ spins in the aromatic residues were assigned by means of two-dimensional (Hβ)Cβ(CγCδ)Hδ and (Hβ)Cβ(CγCδCϵ)Hϵ experiments (25Yamazaki T. Formankay J.D. Kay L.E. 2-Dimensional Nmr experiments for correlating C-13-β and H-1-δ/ϵ chemical-shifts of aromatic residues in C-13-labeled proteins via scalar couplings.J. Am. Chem. Soc. 1993; 115: 11054-11055Crossref Scopus (395) Google Scholar). For detection of intermolecular NOEs, a series of filter-related experiments were conducted, namely a 13C-filtered/13C-edited NOESY using 13C,15N-labeled Csk-SH2 complexed with nonlabeled Cbp5 peptide and a 13C-filtered/13C-edited NOESY and 13C,15N-filtered/15N-edited NOESY using 13C,15N-labeled Cbp5 peptide complexed with nonlabeled Csk-SH2 (26Zwahlen C. Legault P. Vincent S.J. Greenblatt J. Konrat R. Kay L.E. Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: Application to a bacteriophage λN-peptide/boxB RNA complex.J. Am. Chem. Soc. 1997; 119: 6711-6721Crossref Scopus (537) Google Scholar). The NOE peaks were manually assigned using Sparky. Distance restraints were generated according to the assignment of the NOE cross-peaks, and pseudo-atom corrections were applied to the upper bound restraints involving methyl, methylene, and aromatic ring protons as described previously (27Umitsu M. Morishita H. Murata Y. Udaka K. Akutsu H. Yagi T. Ikegami T. 1H, 13C, and 15N resonance assignments of the first cadherin domain of cadherin-related neuronal receptor (CNR)/protocadherin α.J. Biomol. NMR. 2005; 31: 365-366Crossref PubMed Scopus (5) Google Scholar). Torsion angle restraints were derived using TALOS+ (28Shen Y. Delaglio F. Cornilescu G. Bax A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts.J. Biomol. NMR. 2009; 44: 213-223Crossref PubMed Scopus (2008) Google Scholar) with the assigned chemical shifts of 1Hα, 13Cα, 13Cβ, 13CO, and 15N, in reference to the x-ray structure of intact Csk (14Ogawa A. Takayama Y. Sakai H. Chong K.T. Takeuchi S. Nakagawa A. Nada S. Okada M. Tsukihara T. Structure of the carboxyl-terminal Src kinase, Csk.J. Biol. Chem. 2002; 277: 14351-14354Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Hydrogen bond restraints, 2.5–3.3 Å for N-O pairs and 1.8–2.3 Å for H-O pairs, were added only to secondary structural regions as confirmed through the corresponding NOE patterns. One disulfide bond between Cys-122 and Cys-164 was confirmed through characteristic 13Cα and 13Cβ chemical shifts (29Sharma D. Rajarathnam K. C-13 NMR chemical shifts can predict disulfide bond formation.J. Biomol. NMR. 2000; 18: 165-171Crossref PubMed Scopus (250) Google Scholar) and used as a restraint for structure calculations. Structure calculations with torsion angle dynamics were performed using CYANA-2.1 (30Güntert P. Mumenthaler C. Wüthrich K. Torsion angle dynamics for NMR structure calculation with the new program DYANA.J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2553) Google Scholar). A total of 100 structures were calculated with 40,000 steps, and after a root-mean-square deviation (r.m.s.d.) for the backbone atoms had reached 1.0 Å, the r−6 sum averaging method, originally implemented in the CYANA calculations, was applied instead of the pseudo-atom corrections. Finally, the 20 structures with the lowest target functions were selected. Molecular models were prepared using MOLMOL (31Koradi R. Billeter M. Wuthrich K. MOLMOL: a program for display and analysis of macromolecular structures.J. Mol. Graphics. 1996; 14 (29–32): 51-55Crossref PubMed Scopus (6487) Google Scholar) and UCSF Chimera (32Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera–a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (27933) Google Scholar). Hydrogen bonds in the complex structure were defined using UCSF Chimera. An accessible surface area for each residue was calculated using MOLMOL. The electrostatic potential of the complex structure was calculated using Delphi (33Gilson M.K. Sharp K.A. Honig B.H. Calculating the electrostatic potential of molecules in solution–Method and error assessment.J. Comput. Chem. 1988; 9: 327-335Crossref Scopus (1082) Google Scholar). All the amino acid sequence alignments were performed using ClustalW, version 2.0 (34Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. Thompson J.D. Gibson T.J. Higgins D.G. Clustal W and Clustal X version 2.0.Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (22511) Google Scholar). The 15N-spin longitudinal (R1) and transverse (R2) relaxation rates and {1H}–15N steady-state NOE values were acquired on Bruker DRX-500 and AvanceII-800 spectrometers (26Zwahlen C. Legault P. Vincent S.J. Greenblatt J. Konrat R. Kay L.E. Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: Application to a bacteriophage λN-peptide/boxB RNA complex.J. Am. Chem. Soc. 1997; 119: 6711-6721Crossref Scopus (537) Google Scholar). R1 relaxation delays were set at 5, 100, 200, 300, 400, 550, 700, 850, 1000, 1150, and 1300 ms, and R2 relaxation delays were set at 7.2, 36, 72, 108, 144, 180, 216, 252, 288, 324, and 360 ms in corresponding experiments. Single exponential curve fitting was performed using Sparky (35Goddard T.D. Kneller D.G. SPARKY 3. University of California, San Francisco, CA1999Google Scholar). The model-free analysis developed by Lipari and Szabo (36Lipari G. Szabo A. Model-free approach to the interpretation of nuclear magnetic-resonance relaxation in macromolecules. 1. Theory and range of validity.J. Am. Chem. Soc. 1982; 104: 4546-4559Crossref Scopus (3395) Google Scholar), with the isotropic rotational diffusion model assumed, was performed using Tensor2 (37Dosset P. Hus J.C. Blackledge M. Marion D. Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data.J. Biomol. NMR. 2000; 16: 23-28Crossref PubMed Scopus (441) Google Scholar). All NMR data were processed and analyzed using NMRPipe (38Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes.J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11529) Google Scholar) and Sparky, respectively. Chemical exchange associated with the interaction was monitored using 15N-labeled Csk-SH2 mixed with each of the nonlabeled phosphopeptides, Cbp5, Cbp6, and Cbp7, at a molar ratio of 2:1 by two-dimensional 15N zz-exchange spectroscopy (39Farrow N.A. Zhang O. Forman-Kay J.D. Kay L.E. A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium.J. Biomol. NMR. 1994; 4: 727-734Crossref PubMed Scopus (387) Google Scholar, 40Wider G. Neri D. Wüthrich K. Studies of slow conformational equilibria in macromolecules by exchange of heteronuclear longitudinal 2-spin-order in a 2D difference correlation experiment.J. Biomol. NMR. 1991; 1: 93-98Crossref Scopus (69) Google Scholar, 41Wang H. He Y. Kroenke C.D. Kodukula S. Storch J. Palmer A.G. Stark R.E. Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids.Biochemistry. 2002; 41: 5453-5461Crossref PubMed Scopus (22) Google Scholar) on a Bruker Avance-III 950 spectrometer with a cryogenic TCI probe. The spectra were acquired with mixing times, τm, of 0, 20, 50, 100, 200, 350, 550, and 800 ms. For each time point, four peaks observed with intensities denoted by IAA(τm), IBB(τm), IBA(τm), and IAB(τm) are, respectively, governed by Equation 1, lAA(τm)=lAA(0)[pA+pBexp(−kexτm)]exp(−R1τm)lBB(τm)=lBB(0)[pB+pAexp(−kexτm)]exp(−R1τm)lBA(τm)=lAA(0)[pB(1−exp(−kexτm)]exp(−R1τm)lAB(τm)=lBB(0)[pA(1−exp(−kexτm)]exp(−R1τm)(Eq. 1) where pi represents the relative population for the site i; kex represents the sum of the forward, k1, and reverse, koff, kinetic rate constants for interconversion between the sites; and R1 represents the longitudinal relaxation rate of the 15N nucleus in the observed spin system. Note that k1 is an apparent pseudo-first-order rate constant that cor" @default.
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• W2027663241 title "Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains" @default.
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