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• W1994024295 abstract "The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its “active” conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases. The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its “active” conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases. Lyn is a member of the Src family of intracellular membrane-associated tyrosine kinases. While the N terminus of each member is unique, this family shares significant homology in the kinase domain, as well as the SH2/SH3 protein interaction domains. Tyrosine phosphorylation controls the activity of Lyn, and other Src family kinases, in two opposing ways (1Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 2Ingley E. Biochim. Biophys. Acta. 2008; 1784: 56-65Crossref PubMed Scopus (254) Google Scholar). Phosphorylation of the C-terminal tail (pTyr508 in Lyn) inhibits activity through promoting its association with the kinases own SH2 domain. In contrast, phosphorylation of a residue within the activation loop (pTyr397) results in activation of the enzyme. Enzymatic inhibition is also enhanced by the binding of the kinases SH3 domain to a left-handed polyproline type II helix situated between the SH2 and kinase domains (2Ingley E. Biochim. Biophys. Acta. 2008; 1784: 56-65Crossref PubMed Scopus (254) Google Scholar). The SH2 and SH3 domains are also utilized by the enzyme for its activation, as well as inactivation, through specific interactions with proteins containing polyproline and/or phosphotyrosine motifs, thus also allowing specific targeting of the kinases to specific substrates/subcellular compartments (3Pawson T. Nash P. Genes Dev. 2000; 14: 1027-1047PubMed Google Scholar). The involvement of Src family kinases in various signaling cascades including cytokine receptor pathways (4Corey S.J. Anderson S.M. Blood. 1999; 93: 1-14Crossref PubMed Google Scholar, 5Rane S.G. Reddy E.P. Oncogene. 2002; 21: 3334-3358Crossref PubMed Scopus (203) Google Scholar) is gradually being elucidated (6Parsons S.J. Parsons J.T. Oncogene. 2004; 23: 7906-7909Crossref PubMed Scopus (717) Google Scholar). Lyn is expressed in hemopoietic cells of erythroid/myeloid and B lymphoid origin (7Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (115) Google Scholar, 8De Franceschi L. Fumagalli L. Olivieri O. Corrocher R. Lowell C.A. Berton G. J. Clin. Investig. 1997; 99: 220-227Crossref PubMed Scopus (103) Google Scholar, 9Robinson D. Chen H.C. Li D. Yustein J.T. He F. Lin W.C. Hayman M.J. Kung H.J. J. Biochem. Sci. 1998; 5: 93-100Google Scholar), neuronal cells, prostate cells (10Goldenberg-Furmanov M. Stein I. Pikarsky E. Rubin H. Kasem S. Wygoda M. Weinstein I. Reuveni H. Ben-Sasson S.A. Cancer Res. 2004; 64: 1058-1066Crossref PubMed Scopus (91) Google Scholar), colon cells (11Bates R.C. Edwards N.S. Burns G.F. Fisher D.E. Cancer Res. 2001; 61: 5275-5283PubMed Google Scholar), and is involved in the transmission of signals from a number of receptors such as Epo (7Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (115) Google Scholar, 12Tilbrook P.A. Palmer G.A. Bittorf T. McCarthy D.J. Wright M.J. Sarna M.K. Linnekin D. Cull V.S. Williams J.H. Ingley E. Schneider-Mergener J. Krystal G. Klinken S.P. Cancer Res. 2001; 61: 2453-2458PubMed Google Scholar, 13Ingley E. McCarthy D.J. Pore J.R. Sarna M.K. Adenan A.S. Wright M.J. Erber W.N. Tilbrook P.A. Klinken S.P. Oncogene. 2005; 24: 336-343Crossref PubMed Scopus (35) Google Scholar), c-Kit (14Linnekin D. DeBerry C.S. Mou S. J. Biol. Chem. 1997; 272: 27450-27455Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), B cell antigen, and c-Mpl (15Lannutti B.J. Drachman J.G. Blood. 2004; 103: 3736-3743Crossref PubMed Scopus (57) Google Scholar) receptors (4Corey S.J. Anderson S.M. Blood. 1999; 93: 1-14Crossref PubMed Google Scholar). Lyn phosphorylates a number of signaling molecules, including PI 3-kinase, PLCγ2, HS1 (16Ingley E. Sarna M.K. Beaumont J.G. Tilbrook P.A. Tsai S. Takemoto Y. Williams J.H. Klinken S.P. J. Biol. Chem. 2000; 275: 7887-7893Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), Cbp (17Ingley E. Schneider J.R. Payne C.J. McCarthy D.J. Harder K.W. Hibbs M.L. Klinken S.P. J. Biol. Chem. 2006; 281: 31920-31929Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), STAT5 (18Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar), and MAP kinase, and Lyn-/- mice display severe defects in the immune system (19Hibbs M.L. Tarlinton D.M. Armes J. Grail D. Hodgson G. Maglitto R. Stacker S.A. Dunn A.R. Cell. 1995; 83: 301-311Abstract Full Text PDF PubMed Scopus (628) Google Scholar) and the erythroid compartment (13Ingley E. McCarthy D.J. Pore J.R. Sarna M.K. Adenan A.S. Wright M.J. Erber W.N. Tilbrook P.A. Klinken S.P. Oncogene. 2005; 24: 336-343Crossref PubMed Scopus (35) Google Scholar, 20Harder K.W. Quilici C. Naik E. Inglese M. Kountouri N. Turner A. Zlatic K. Tarlinton D.M. Hibbs M.L. Blood. 2004; 104: 3901-3910Crossref PubMed Scopus (81) Google Scholar). An important role for Lyn in leukemia has been suggested by several studies (21Roginskaya V. Zuo S. Caudell E. Nambudiri G. Kraker A.J. Corey S.J. Leukaemia. 1999; 13: 855-861Crossref PubMed Scopus (85) Google Scholar, 22Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (594) Google Scholar, 23Golas J.M. Arndt K. Etienne C. Lucas J. Nardin D. Gibbons J. Frost P. Ye F. Boschelli D.H. Boschelli F. Cancer Res. 2003; 63: 375-381PubMed Google Scholar, 24Warmuth M. Simon N. Mitina O. Mathes R. Fabbro D. Manley P.W. Buchdunger E. Forster K. Moarefi I. Hallek M. Blood. 2003; 101: 664-672Crossref PubMed Scopus (121) Google Scholar, 25Wilson M.B. Schreiner S.J. Choi H.J. Kamens J. Smithgall T.E. Oncogene. 2002; 21: 8075-8088Crossref PubMed Scopus (119) Google Scholar). Primary acute myeloid leukemia (AML) cells display elevated Lyn kinase activity (21Roginskaya V. Zuo S. Caudell E. Nambudiri G. Kraker A.J. Corey S.J. Leukaemia. 1999; 13: 855-861Crossref PubMed Scopus (85) Google Scholar) and Lyn is critical for maintaining AML cell proliferation and anti-apoptotic pathways (26Dos Santos C. Demur C. Bardet V. Prade-Houdellier N. Payrastre B. Recher C. Blood. 2008; 111: 2269-2279Crossref PubMed Scopus (127) Google Scholar). While the BCR-Abl fusion protein is the initiating molecule for chromic myeloid leukemia (CML), there is a crucial downstream role for Lyn in BCR-Abl-induced leukemogenesis (23Golas J.M. Arndt K. Etienne C. Lucas J. Nardin D. Gibbons J. Frost P. Ye F. Boschelli D.H. Boschelli F. Cancer Res. 2003; 63: 375-381PubMed Google Scholar, 24Warmuth M. Simon N. Mitina O. Mathes R. Fabbro D. Manley P.W. Buchdunger E. Forster K. Moarefi I. Hallek M. Blood. 2003; 101: 664-672Crossref PubMed Scopus (121) Google Scholar, 25Wilson M.B. Schreiner S.J. Choi H.J. Kamens J. Smithgall T.E. Oncogene. 2002; 21: 8075-8088Crossref PubMed Scopus (119) Google Scholar). There is a direct link between Lyn and BCR-Abl signaling pathways as Lyn phosphorylates Tyr177 of BCR-Abl (27Wu J. Meng F. Lu H. Kong L. Bornmann W. Peng Z. Talpaz M. Donato N.J. Blood. 2008; 111: 3821-3829Crossref PubMed Scopus (98) Google Scholar, 28Meyn III, M.A. Wilson M.B. Abdi F.A. Fahey N. Schiavone A.P. Wu J. Hochrein J.M. Engen J.R. Smithgall T.E. J. Biol. Chem. 2006; 281: 30907-30916Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), thus recruiting the adaptor Gab2, both of which are essential for BCR-Abl oncogenesis (29Pendergast A.M. Quilliam L.A. Cripe L.D. Bassing C.H. Dai Z. Li N. Batzer A. Rabun K.M. Der C.J. Schlessinger J. Gishizky M.L. Cell. 1993; 75: 175-185Abstract Full Text PDF PubMed Scopus (592) Google Scholar). Significantly, Imatinib-resistant CML cells have elevated Lyn levels and kinase activity (22Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (594) Google Scholar). Further, ablation of Lyn from Imatinib-resistant CML cells resulted in the induction of apoptosis (30Ptasznik A. Nakata Y. Kalota A. Emerson S.G. Gewirtz A.M. Nat. Med. 2004; 10: 1187-1189Crossref PubMed Scopus (198) Google Scholar). A significant role for Lyn in the development of certain solid tumors has also come to light. Colon carcinoma cells utilize Lyn in the activation of the Akt anti-apoptotic pathway, and drug-resistant cells show elevated Lyn kinase activity (11Bates R.C. Edwards N.S. Burns G.F. Fisher D.E. Cancer Res. 2001; 61: 5275-5283PubMed Google Scholar). Lyn is also involved in the signaling mechanisms regulating prostate cancer cells (31Sumitomo M. Shen R. Walburg M. Dai J. Geng Y. Navarro D. Boileau G. Papandreou C.N. Giancotti F.G. Knudsen B. Nanus D.M. J. Clin. Investig. 2000; 106: 1399-1407Crossref PubMed Scopus (124) Google Scholar). Significantly, inhibition of Lyn in prostate cancer cell lines resulted in reduced proliferation in vitro and in prostatic cancer xenograft models (10Goldenberg-Furmanov M. Stein I. Pikarsky E. Rubin H. Kasem S. Wygoda M. Weinstein I. Reuveni H. Ben-Sasson S.A. Cancer Res. 2004; 64: 1058-1066Crossref PubMed Scopus (91) Google Scholar). Thus several lines of evidence point to a significant involvement of Lyn in both leukemic and solid tumor development. The Src-family as well as other cytoplasmic (e.g. Abl) and receptor (e.g. EGF-R) tyrosine kinases are important targets or therapeutic intervention (32Druker B.J. Talpaz M. Resta D.J. Peng B. Buchdunger E. Ford J.M. Lydon N.B. Kantarjian H. Capdeville R. Ohno-Jones S. Sawyers C.L. N. Engl. J. Med. 2001; 344: 1031-1037Crossref PubMed Scopus (4457) Google Scholar, 33Talpaz M. Shah N.P. Kantarjian H. Donato N. Nicoll J. Paquette R. Cortes J. O'Brien S. Nicaise C. Bleickardt E. Blackwood-Chirchir M.A. Iyer V. Chen T.T. Huang F. Decillis A.P. Sawyers C.L. N. Engl. J. Med. 2006; 354: 2531-2541Crossref PubMed Scopus (1477) Google Scholar). Several specific (e.g. Imatinib) and some less specific (e.g. Dasatinib) small molecules have been generated that mostly act as ATP competitive inhibitors and have been successfully employed for leukemia/cancer treatment in the clinic (32Druker B.J. Talpaz M. Resta D.J. Peng B. Buchdunger E. Ford J.M. Lydon N.B. Kantarjian H. Capdeville R. Ohno-Jones S. Sawyers C.L. N. Engl. J. Med. 2001; 344: 1031-1037Crossref PubMed Scopus (4457) Google Scholar, 33Talpaz M. Shah N.P. Kantarjian H. Donato N. Nicoll J. Paquette R. Cortes J. O'Brien S. Nicaise C. Bleickardt E. Blackwood-Chirchir M.A. Iyer V. Chen T.T. Huang F. Decillis A.P. Sawyers C.L. N. Engl. J. Med. 2006; 354: 2531-2541Crossref PubMed Scopus (1477) Google Scholar). Crystal structures of Src (1Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 34Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1249) Google Scholar), Hck (35Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1045) Google Scholar, 36Schindler T. Sicheri F. Pico A. Gazit A. Levitzki A. Kuriyan J. Mol. Cell. 1999; 3: 639-648Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar), and Lck (37Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (423) Google Scholar, 38Zhu X. Kim J.L. Newcomb J.R. Rose P. E, Stover D.R. Toledo L.M. Zhao H. Morgenstern K.A. Structure. 1999; 7: 651-661Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) have enabled a detailed investigation of how the Src family of kinases are regulated, and the way in which small molecule inhibitors can inactivate these enzymes. To extend our understanding, and provide a structural framework for the design of specific inhibitors to the Src family of kinases, we have determined the crystal structures of the kinase domain of Lyn in complex with 3 different inhibitors, AMP-PNP, 2The abbreviations used are: AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; PTK, protein-tyrosine kinase domain; VDW, van der Waals; DTT, dithiothreitol; r.m.s.d., root mean square deviation. PP2, and Dasatinib, as well as the non-liganded form of the enzyme, at 2.5–2.7-Å resolution. Plasmid Construction—All plasmid constructs were generated by site-directed mutagenesis using oligonucleotides and subcloned in-frame into the appropriate vector and confirmed by sequencing. The murine Lyn kinase domain (amino acids 239–512) was subcloned in to the baculovirus expression vector pFastBacHTA (Invitrogen) generating a His6-tagged fusion resulting in a 5-amino acid N-terminal extension (GAMDP) to the Lyn kinase domain after tobacco etch virus (TEV) cleavage. The murine Csk-binding protein (Cbp, amino acids 74–474) was subcloned into pET44a (Merck, Darmstadt, Germany) to be expressed as a NusA-His6-tagged fusion in bacteria (Rosetta2, Merck), with replacement of the thrombin with a TEV cleave site using site-directed mutagenesis, for expression and purification using the Profinia system (Bio-Rad) for use as a biologically significant substrate for Lyn kinase assays. Protein Expression and Purification of Lyn Kinase Domain—Recombinant bacmid DNA was isolated and used to transfect Spodoptera frugiperda (Sf9) insect cells. Baculovirus obtained from the transfection culture was used to infect Sf9 cells grown in suspension to a density of 2 × 106 cells per ml, at a multiplicity of infection greater than 10, and harvested 48 h after infection. Cells were resuspended in a buffer consisting of 20 mm Tris HCl, pH 8.0, 500 mm NaCl, 5% glycerol, 3 mm β-mercaptoethanol, 0.1% thesit, supplemented with complete protease inhibitors mixture (Roche Applied Sciences), lysed by sonication and centrifuged at 45,000 × g for 1 h at 4 °C. The supernatant was filtered and loaded onto ProBond nickel-chelating resin (Invitrogen). After extensive washing, the recombinant protein was eluted with buffer plus 100–300 mm imidazole and Lyn-containing fractions were pooled. The His6 tag was removed by treatment with TEV protease during overnight dialysis against 20 mm Tris HCl, pH 8.0, 25 mm NaCl, 5% glycerol, 2 mm DTT, 0.5 mm EDTA, at 4 °C. The digested protein was bound to a HiTrap Q column (GE Healthcare) equilibrated in the same buffer and eluted with a NaCl gradient. Lyn-containing fractions were pooled, concentrated to 2.5 ml and loaded onto a Superdex 75 gel filtration column (HiLoad 16/60; GE Healthcare) equilibrated in 20 mm Tris-HCl, pH 8.0, 200 mm NaCl, 2 mm DTT, 0.5 mm EDTA. Purified Lyn kinase domain was concentrated to 8 mg/ml for crystallization. Protein Analysis and Western Blotting—Mass spectrometric analysis of purified Lyn protein before and after auto-kinase assays were performed by Proteomics International (East Perth, WA, Australia) on protein gel plugs that were destained, trypsin-digested, and peptides extracted according to standard techniques (39Bringans S. Eriksen S. Kendrick T. Gopalakrishnakone P. Livk A. Lock R. Lipscombe R. Proteomics. 2008; 8: 1081-1096Crossref PubMed Scopus (149) Google Scholar). Peptides were analyzed by electrospray ionization mass spectrometry (LC/MS) using the Ultimate 3000 HPLC system (Dionex, Sunnyvale, CA) coupled to a Q TRAP 4000 mass spectrometer (Applied Biosystems, Foster City, CA). Tryptic peptides were loaded onto a C18PepMap100 reversed phase column (Dionex) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v) at a flow rate of 300 nl/min. Spectra were analyzed to identify proteins of interest using Mascot sequence matching software (Matrix Science, London, UK) with the data base and taxonomy set as Ludwig NR and All Taxonomy, respectively. The phosphorylation detection was achieved with precursor ion scanning for a loss of 79 mass units in negative mode (PO3-) and subsequent MSMS fragmentation of those selected peptides to determine their identity. Protein concentration was estimated using the Bio-Rad Dc protein assay according to the manufacturer’s instructions, using bovine serum albumin as a standard. Exo-kinase kinase assays were performed essentially as previously described (7Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (115) Google Scholar), with minor modifications, using NusA-Cbp (5 mg/ml) as substrate in 50 mm Tris-HCl, pH 7.4, 1 mm DTT, 10 mm MgCl2, 50 μm ATP, at 30 °C. Kinase inhibitors Dasatinib (Bristol-Myers-Squibb, New York, NY), PP2 (Merck) and SU6656 (Merck) were added to reaction mixtures 10 min prior to the addition of ATP. Auto-kinase reactions were undertaken under identical conditions to those described for the exo-kinase assays, only no NusA-Cbp substrate was added. Reactions contained 0.1 μg/10 μl Lyn kinase and were stopped by the addition of SDS loading buffer. Reactions were then analyzed by Western blotting for phosphotyrosine incorporation into the Cbp substrate. Western blotting was performed essentially as described previously (7Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (115) Google Scholar, 40Ingley E. Chappell D. Poon S.Y. Sarna M.K. Beaumont J.G. Williams J.H. Stillitano J.P. Tsai S. Leedman P.J. Tilbrook P.A. Klinken S.P. J. Biol. Chem. 2001; 276: 43428-43434Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Antibodies used included anti-phosphotyrosine 4G10-HRP (Millipore, Billerica, MA) and anti-phospho-Src (pY416) (Cell Signaling Technology, Danvers, MA). Secondary antibodies were coupled to horseradish peroxidase (Amersham, Buckinghamshire, UK) and detected by enhanced chemiluminescence (Amersham Biosciences). Western blots were quantitated using a ChemDoc XRS (Bio-Rad) and Quantity One (v4.5.2, Bio-Rad). Crystallization of Lyn Kinase Domain and Formation of Inhibitor Complexes—Crystals were grown at 4 °C using the hanging-drop vapor-diffusion method. Purified Lyn kinase domain was mixed with an equal volume of a reservoir solution containing 23% polyethylene glycol 3350, 0.1 m NaCl, and 0.1 m Na.Hepes, pH 7.5. Crystals formed after 1–3 days. Inhibitors PP2 (1-tert-butyl-3-(4-chlorophenyl)-2H-pyrazolo[4, 5-e]pyrimidin-1-ium-4-amine) and Dasatinib (BMS-354825; N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide) in DMSO at 10 mm, or diluted 10-fold in reservoir solution, were added to drops containing Lyn crystals, and incubated at 4 °C for 1–2 h. AMP-PNP and MgCl2 at 50 mm each were added to crystal-containing drops and incubated for up to 3 weeks at 4 °C. X-Ray Data Collection, Structure Determination, and Refinement—Crystals were flash-frozen using the reservoir solution plus 10% glycerol as a cryoprotectant. Data sets at 2.5–2.76-Å resolution were merged and processed using MOSFLM and SCALA in the CCP4i suite. The crystals, with unit cell dimensions a = b = 127.3 Å and c = 53.8 Å, belong to space group H3, with one monomer in the asymmetric unit. The apo-structure was solved by molecular replacement using the program PHASER in the CCP4i suite. Lck was used as a search model (Protein Data Bank ID: 1QPD) with all non-conserved residues substituted with alanine. The structure was refined using the CCP4i suite program REFMAC5 by rigid body fitting followed by restrained refinement interspersed with rounds of model building with the program COOT. For inhibitor complex structures, difference Fourier analyses were used to evaluate ligand binding. Accordingly, unbiased features in electron density maps revealed the location of inhibitor molecules, and the subsequent structures were refined as listed above. For refinement of the inhibitor complexes, the same Rfree data set as selected in the apo crystal form. A summary of the data collection and refinement statistics is presented in Table 1.TABLE 1Data collection and refinement statisticsApo-LynLyn-PP2Lyn-DasatinibLyn-AMP-PNPTemperature100K100K100K100KSpace groupH3H3H3H3Cell dimensions (Å) (a = b, c)127.31, 53.84128.47, 54.17127.38, 55.62127.24, 54.88Resolution limits (Å)aThe value in parentheses is for the highest resolution bin (approximate interval, 0.1 Å)63.3-2.5 (2.64-2.5)48.7-2.76 (2.91-2.76)39.2-2.6 (2.74-2.6)38.9-2.7 (2.85-2.7)No. observations28576166742462118056No. unique reflections112598337101069082Completeness (%)aThe value in parentheses is for the highest resolution bin (approximate interval, 0.1 Å)100 (100)97.1 (83.0)97.6 (95.7)99.8 (100)Rmerge (%)aThe value in parentheses is for the highest resolution bin (approximate interval, 0.1 Å),bRmerge = Σ|Ihkl-<Ihkl>|/ΣIhkl3.7 (35.3)4.3 (33.8)5.4 (35.4)4.3 (34.1)I/σIaThe value in parentheses is for the highest resolution bin (approximate interval, 0.1 Å)21.1 (2.2)16.3 (2.0)16.6 (2.3)17.1 (2.1)Multiplicity2.52.02.42.0R-factor (%)cRfactor = ΣIhkl½|Fo – |Fc½½|/ΣIhkl|Fo| for all data except 5%, which was used for the Rfree calculation20.519.519.819.9R-free (%)cRfactor = ΣIhkl½|Fo – |Fc½½|/ΣIhkl|Fo| for all data except 5%, which was used for the Rfree calculation23.823.123.124.5Number of atoms:-Protein2044203120812044-Ligand213331-Water14163815Ramachandran plot, most favored %94909594R.m.s.d. from idealityBond lengths (Å)0.0070.0080.0070.011Bond angles1.0351.2351.2041.153a The value in parentheses is for the highest resolution bin (approximate interval, 0.1 Å)b Rmerge = Σ|Ihkl-<Ihkl>|/ΣIhklc Rfactor = ΣIhkl½|Fo – |Fc½½|/ΣIhkl|Fo| for all data except 5%, which was used for the Rfree calculation Open table in a new tab Biochemical Analysis of Purified Lyn Kinase Domain—The purified kinase domain of Lyn was assessed for post-translational modifications by Western blot and mass spectrometry analysis. Using an antibody specific for the common activation loop phosphorylation site found in Src family kinases (pTyr416 in Src, pTyr397 in Lyn), only after auto-activation was significant reactivity observed (Fig. 1A). This was confirmed by mass spectrometric analysis, showing the purified kinase domain was essentially unphosphorylated at this site, but upon incubation with Mg2+-ATP it became rapidly phosphorylated. The enzyme was also able to rapidly phosphorylate the substrate Cbp in exo-kinase assays showing the capacity of the purified protein to display functional activity (Fig. 1B). Further, the ability of small molecules to inhibit the enzymes ability to phosphorylate Cbp were analyzed, showing Dasatinib (IC50 11 nm), PP2 (IC50 9 nm), and SU6656 (IC50 35 nm) could all strongly inhibit the activity of Lyn (Fig. 1C). The PP2 and Dasatinib inhibitors were then used to examine the structural basis of binding and specificity by x-ray crystallography. Structure of the Lyn Kinase Domain—To determine the overall atomic architecture of the Lyn PTK, and provide a baseline for structural comparison of inhibitor binding to Lyn, the kinase domain of murine Lyn was crystallized in the absence of inhibitor and its structure determined at 2.5-Å resolution and refined to an Rfact = 20.5% and Rfree = 23.8%. The refined model, comprising residues 239–501, is typical of the bi-lobal protein-tyrosine kinase fold and closely resembles the structures of Src, Lck, and Hck (Fig. 2A). Briefly, the N-terminal lobe of the PTK consists of a 5-stranded anti-parallel twisted β-sheet and a single large α-helix, termed the αC helix. The C-terminal lobe is larger and predominantly helical, but includes a 2-stranded anti-parallel β-sheet. Between the two lobes is a deep cleft that forms the ATP-binding pocket, and connecting them is a loop that borders the cleft and forms a hinge that confers flexibility to the overall structure, allowing for relative movement of the lobes. The catalytic activity of protein tyrosine kinase can be regulated by the phosphorylation state of the activation loop Tyr residue (Tyr397 in Lyn) and is correlated with movements in the loop and αC. The region of the activation loop spanning this residue is thus highly flexible, and in the apo Lyn structure residues 393–399, inclusive, are disordered and not included in the model, thus not revealing the phosphorylation state of Tyr397, although Western blot and mass spectrometric analyses clearly indicated that the Lyn PTK was unphosphorylated. Structural comparison with unphosphorylated, inactive Src and Hck, and active, phosphorylated Lck (Fig. 2B) shows that despite the lack of phosphorylation in Lyn PTK, the activation loop and αC are in the active conformations. AMP-PNP Binding to Lyn—To provide a model for the substrate-bound state of the enzyme, Lyn crystals were soaked with the non-hydrolyzable ATP analogue adenyl imidodiphosphate (AMP-PNP) in the presence of Mg2+ ions and the resulting complex was solved at 2.7 Å resolution and refined to an Rfact = 19.9% and Rfree = 24.5%. As expected, AMP-PNP binds in the cleft between the two lobes of the kinase domain (Fig. 3A forming extensive van der Waals (VDW) and hydrogen-bonding interactions. The planar adenine ring is sandwiched between the hydrophobic residues of the N-terminal lobe (Leu253, Val261, and Ala273), the hinge region (Phe321 and Met322) and the C-terminal lobe (Leu374). In addition, two hydrogen bonds link the adenine group through N6 and N1 to the hinge residues Glu320O and Met322N, respectively, as seen with AMP-PNP-binding to other Src family kinases. In the published complexes of AMP-PNP with Src (1Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar) and Lck (38Zhu X. Kim J.L. Newcomb J.R. Rose P. E, Stover D.R. Toledo L.M. Zhao H. Morgenstern K.A. Structure. 1999; 7: 651-661Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), the ribose group is hydrogen-bonded to the Ser residue conserved in all but one Src-family PTKs (326 in Lyn) either through a water-mediated hydrogen bond in Src, or directly in the case of Lck where the side chain points toward the ligand. In the Lyn structure, as in Src, Ser326 is pointing away from the ribose group, but at the resolution of this Lyn structure, no hydrogen-bonded water molecule is detected. Asp385 of the conserved DFG motif forms hydrogen bonds with the α- and β-phosphates and a water-mediated hydrogen bond to the imido-nitrogen. The β-phosphate group interacts with the glycine loop residues Ala255 and Gly259 via a water molecule that is accommodated by a small shift in the loop location with respect to the apo structure. A third water molecule links Arg369 to the γ-phosphate. Thus, extensive contacts and hydrogen bonds anchor the substrate analogue and poise the tri-phosphate group for catalysis. PP2 Binding to Lyn—For insight into the structural basis of small molecule inhibition of Lyn PTK by the pan-Src kinase inhibitor PP2, the PP2-Lyn complex was formed by soaking apo Lyn crystals with PP2, and the structure was solved at 2.76-Å resolution and refined to an Rfact = 19.5% and Rfree = 23.1% (Fig. 3B). PP2 binds to Lyn kinase in a very similar position to AMP-PNP, with the respective purine rings almost co-planar. The two adenine group hydrogen bonds to residues Glu320 and Met322 are prese" @default.
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• W1994024295 title "Crystal Structures of the Lyn Protein Tyrosine Kinase Domain in Its Apo- and Inhibitor-bound State" @default.
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