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• W2022742531 abstract "Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes. Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes. The Drosophila disabled gene product (DAB) 1The abbreviations used are: DAB, the Drosophila disabled gene product; DH, DAB homology; Dab1-DH, the DH domain of Dab1; Dab2-DH, the DH domain of Dab2; NPXY, Asp-Pro-X-Try; APP, amyloid precursor protein; Ins-1,4,5-P3, inositol 1,4,5-trisphosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-diphosphate; PTB/PI, phosphotyrosine binding/phosphotyrosine interaction; apoER2, apolipoprotein E receptor type 2; PH, Pleckstrin homology; HSQC, heteronuclear single quantum coherence spectroscopy; BSA, bovine serum albumin; r.m.s.d., root mean square deviation.1The abbreviations used are: DAB, the Drosophila disabled gene product; DH, DAB homology; Dab1-DH, the DH domain of Dab1; Dab2-DH, the DH domain of Dab2; NPXY, Asp-Pro-X-Try; APP, amyloid precursor protein; Ins-1,4,5-P3, inositol 1,4,5-trisphosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-diphosphate; PTB/PI, phosphotyrosine binding/phosphotyrosine interaction; apoER2, apolipoprotein E receptor type 2; PH, Pleckstrin homology; HSQC, heteronuclear single quantum coherence spectroscopy; BSA, bovine serum albumin; r.m.s.d., root mean square deviation. was identified as the result of a genetic screen designed to isolate modifier genes of the abl tyrosine kinase (1Bennett R.L. Hoffmann F.M. Development. 1992; 116: 953-966PubMed Google Scholar, 2Gertler F.B. Hill K.K. Clark M.J. Hoffmann F.M. Genes Dev. 1993; 7: 441-453Crossref PubMed Scopus (112) Google Scholar, 3Gertler F.B. Bennett R.L. Clark M.J. Hoffmann F.M. Cell. 1989; 58: 103-113Abstract Full Text PDF PubMed Scopus (199) Google Scholar). Defects in abl and disabled prevent the formation of proper axonal connections in the central nervous system and cause the death of flies during embryonic development. DAB is tyrosine-phosphorylated by the sevenless receptor kinase and functions as an adaptor protein to recruit SH2-SH3 domain proteins to the signaling complex in Drosophila (4Le N. Simon M.A. Mol. Cell. Biol. 1998; 18: 4844-4854Crossref PubMed Scopus (26) Google Scholar). An evolutionarily conserved family of disabled proteins was revealed by the discovery of a 96-kDa growth factor-responsive phosphoprotein possessing an amino-terminal region of ∼150 amino acids that is highly similar to the amino terminus of DAB (5Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). DAB family members contain an amino-terminal DAB homology (DH) domain that acts as a protein-protein and protein-phospholipid interaction module and is fused to diverse carboxyl-terminal sequences. The DH domain has been identified in two mammalian proteins, Dab1 (also called mDab) and Dab2 (also known as p96 or Doc-2) (5Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 6Howell B.W. Gertler F.B. Cooper J.A. EMBO J. 1997; 16: 121-132Crossref PubMed Scopus (298) Google Scholar). Dab1 plays a key role in migration of neurons during brain development as a component of the Reelin signaling pathway (7Rice D.S. Curran T. Annu. Rev. Neurosci. 2001; 24: 1005-1039Crossref PubMed Scopus (573) Google Scholar, 8Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273,: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 9D'Arcangelo G. Homayouni R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 10Curran T. D'Arcangelo G. Brain Res. Rev. 1998; 26: 285-294Crossref PubMed Scopus (211) Google Scholar, 11Lambert D.R. Goffinet A.M. Adv. Anat. Embryol. Cell Biol. 1998; 150: 1-106Crossref PubMed Google Scholar, 12Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (772) Google Scholar, 13Rice D.S. Sheldon M. D'Arcangelo G. Nakajima K. Goldowitz D. Curran T. Development. 1998; 125: 3719-3729Crossref PubMed Google Scholar, 14Howell B.W. Herrick T.M. Cooper J.A. Genes Dev. 1999; 13: 643-648Crossref PubMed Scopus (353) Google Scholar). Reelin, a large glycoprotein secreted by pioneer cell populations early in development (10Curran T. D'Arcangelo G. Brain Res. Rev. 1998; 26: 285-294Crossref PubMed Scopus (211) Google Scholar, 11Lambert D.R. Goffinet A.M. Adv. Anat. Embryol. Cell Biol. 1998; 150: 1-106Crossref PubMed Google Scholar), directs the positioning of neurons during brain development by binding to very low density lipoprotein receptors or apolipoprotein E type 2 receptors (apoER2) (9D'Arcangelo G. Homayouni R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 12Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (772) Google Scholar). Engagement of these receptors triggers tyrosine phosphorylation of Dab1 (9D'Arcangelo G. Homayouni R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 13Rice D.S. Sheldon M. D'Arcangelo G. Nakajima K. Goldowitz D. Curran T. Development. 1998; 125: 3719-3729Crossref PubMed Google Scholar, 14Howell B.W. Herrick T.M. Cooper J.A. Genes Dev. 1999; 13: 643-648Crossref PubMed Scopus (353) Google Scholar, 15Benhayon D. Magdaleno S. Curran T. Brain Res. Mol. Brain Res. 2003; 112: 33-45Crossref PubMed Scopus (89) Google Scholar), creating a scaffold that recruits signaling proteins possessing SH2 domains. Fyn, an Src tyrosine kinase family member, is directly involved in Reelin-induced Dab1 phosphorylation (16Arnaud L. Ballif B.A. Forster E. Cooper J.A. Curr. Biol. 2003; 13: 9-17Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 17Bock H.H. Herz J. Curr. Biol. 2003; 13: 18-26Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Dab1 is also known to bind to the amyloid precursor family of proteins (18Homayouni R. Rice D.S. Sheldon M. Curran T. J. Neurosci. 1999; 19: 7507-7515Crossref PubMed Google Scholar, 19Howell B.W. Lanier L.M. Frank R. Gertler F.B. Cooper J.A. Mol. Cell. Biol. 1999; 19: 5179-5188Crossref PubMed Scopus (328) Google Scholar), to a novel protocadherin (20Homayouni R. Rice D.S. Curran T. Biochem. Biophys. Res. Commun. 2001; 289: 539-547Crossref PubMed Scopus (48) Google Scholar, 21Senzaki K. Ogawa M. Yagi T. Cell. 1999; 99: 635-647Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), and to integrin β (22Dulabon L. Olson E.C. Taglienti M.G. Eisenhuth S. McGrath B. Walsh C.A. Kreidberg J.A. Anton E.S. Neuron. 2000; 27: 33-44Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar), although no direct connection between these interactions and specific signaling events has been established. Dab2 participates in a distinct subset of receptor-mediated events. The requirement of Dab2 for visceral endoderm development (23Morris S.M. Tallquist M.D. Rock C.O. Cooper J.A. EMBO J. 2002; 21: 1555-1564Crossref PubMed Scopus (170) Google Scholar) is consistent with its proposed function as an adapter in the transforming growth factor transforming growth factor-β/SMAD2 pathway (24Hocevar B.A. Smine A. Xu X.X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (199) Google Scholar). Ablation of Dab2 results in reduced numbers of clathrin-coated pits in kidney proximal tubule cells and the secretion of plasma proteins into the urine (23Morris S.M. Tallquist M.D. Rock C.O. Cooper J.A. EMBO J. 2002; 21: 1555-1564Crossref PubMed Scopus (170) Google Scholar). This phenotype is consistent with the formation of Dab2-megalin complexes (25Oleinikov A.V. Zhao J. Makker S.P. Biochem. J. 2000; 347: 613-621Crossref PubMed Scopus (128) Google Scholar) and the binding of Dab2 to clathrin-coated pits (26Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (248) Google Scholar). In addition, Dab2 specifically associates with PtdIns-4,5-P2, clathrin (26Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (248) Google Scholar, 27Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar), and cytoskeletal components (28Morris S.M. Arden S.D. Roberts R.C. Kendrick-Jones J. Cooper J.A. Luzio J.P. Buss F. Traffic. 2002; 3: 331-341Crossref PubMed Scopus (196) Google Scholar, 29Rosenbauer F. Kallies A. Scheller M. Knobeloch K.P. Rock C.O. Schwieger M. Stocking C. Horak I. EMBO J. 2002; 21: 211-220Crossref PubMed Scopus (55) Google Scholar) and, therefore, may participate in both endocytic trafficking of lipoprotein receptors and cell adhesion/spreading. The DH domain belongs to the Pleckstrin homology (PH) superfamily of structures (30Blomberg N. Baraldi E. Nilges M. Saraste M. Trends Biochem. Sci. 1999; 24: 441-445Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and it most closely resembles the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domains present in many proteins involved in protein-protein interaction and signal transduction (31Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 32Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (106) Google Scholar, 33Borg J.P. Margolis B. Curr. Top. Microbiol. Immunol. 1998; 228: 23-38PubMed Google Scholar, 34Yan K.S. Kuti M. Zhou M.M. FEBS Lett. 2002; 513: 67-70Crossref PubMed Scopus (60) Google Scholar). The PTB/PI domains interact with target proteins and lipids; their binding specificities are crucial to specific functions revealed by high resolution structural studies (32Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (106) Google Scholar, 33Borg J.P. Margolis B. Curr. Top. Microbiol. Immunol. 1998; 228: 23-38PubMed Google Scholar, 34Yan K.S. Kuti M. Zhou M.M. FEBS Lett. 2002; 513: 67-70Crossref PubMed Scopus (60) Google Scholar). There are three functional groups of NPXY binding PTB/PI domains (32Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (106) Google Scholar). One group, typified by Shc and IRS-1, interacts with NPXpY motifs (pY is phosphotyrosine). The second group is defined by the DH domain, which binds only to the unphosphorylated NPXY sequence (8Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273,: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 27Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar). In the third group, the PTB/PI domains of X-11 and Fe65 bind to NPXY motifs independent of their phosphorylation status (32Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (106) Google Scholar). The Shc PTB/PI domain also binds to phosphoinositides at a site that overlaps with the peptide binding region (35Ravichandran K.S. Zhou M.M. Pratt J.C. Harlan J.E. Walk S.F. Fesik S.W. Burakoff S.J. Mol. Cell. Biol. 1997; 17: 5540-5549Crossref PubMed Google Scholar), whereas the DH domain preferentially binds PtdIns-4,5-P2 independent of the protein interaction site (19Howell B.W. Lanier L.M. Frank R. Gertler F.B. Cooper J.A. Mol. Cell. Biol. 1999; 19: 5179-5188Crossref PubMed Scopus (328) Google Scholar). The structures of four NPXY-binding PTB/PI domains are known: Shc, IRS-1, SNT, and X-11 (36Zhou M.M. Ravichandran K.S. Olejniczak E.F. Petros A.M. Meadows R.P. Sattler M. Harlan J.E. Wade W.S. Burakoff S.J. Fesik S.W. Nature. 1995; 378: 584-592Crossref PubMed Scopus (323) Google Scholar, 37Eck M.J. Dhe-Paganon S. Trub T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 38Dhalluin C. Yan K. Plotnikova O. Lee K.W. Zeng L. Kuti M. Mujtaba S. Goldfarb M.P. Zhou M.M. Mol. Cell. 2000; 6: 921-929Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 39Zhang Z. Lee C.H. Mandiyan V. Borg J.P. Margolis B. Schlessinger J. Kuriyan J. EMBO J. 1997; 16: 6141-6150Crossref PubMed Scopus (138) Google Scholar). All contain a PH domain-like fold (30Blomberg N. Baraldi E. Nilges M. Saraste M. Trends Biochem. Sci. 1999; 24: 441-445Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) consisting of a seven-stranded β sandwich flanked by a carboxyl-terminal helix. The Shc, IRS-1, and SNT structures illustrate specific binding modes for NPXpY sequences, whereas the PTB/PI domain structure of X-11 explains its ability to bind both phosphorylated and unphosphorylated peptides. We have investigated the structures of the DH domains of Dab1 and Dab2 to elucidate the structural basis for recognition by the DH domain of the unphosphorylated NPXY sequence motif and PtdIns-P2. We report three structures, the Dab2-DH structures in the ligand-free state and the binary complex with the NPXY peptide of APP and the Dab1-DH structure in the ternary complex with the APP peptide and Ins-1,4,5-P3. The Dab2-DH structures are the first high resolution model of Dab2, showing the structural similarity to Dab1-DH. We present a comparative analysis of the peptide and the phosphoinositide binding pockets of Dab1 and Dab2. While this manuscript was in preparation, the ternary complex structure of Dab1-DH with the apoER2 peptide and Ins-1,4,5-P3 was reported (40Stolt P.C. Jeon H. Song H.K. Herz J. Eck M.J. Blacklow S.C. Structure (Lond.). 2003; 11: 569-579Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). This recent ternary complex structure is very similar to our ternary complex structure in that it shows two independent binding sites for the peptide and phosphoinositide. A major difference between the two ternary complex structures is found at the orientation of Ins-1,4,5-P3 in the binding site. The recent ternary complex structure, which was solved using crystals obtained by soaking with PtdIns-4,5-P2, describes the binding of Ins-1,4,5-P3 in two different orientations. In contrast, our ternary complex structure, which was solved using crystals obtained by co-crystallizing with Ins-1,4,5-P3, resolves the uncertainty about the orientation of Ins-1,4,5-P3 in the binding site. Taken together these structural insights clarify the protein and phospholipid recognition modes that are crucial to the regulation of biological processes by the DH-domain proteins. Crystallography—We cloned cDNAs encoding Dab1-DH (residues 25–183) and Dab2-DH (residues 33–191) into a pET bacterial overexpression vector (Novagen). The proteins were expressed in Escherichia coli BL21(DE3) cells and purified by three column steps (SP-Sepharose, resource S, and Superdex 75 column;, all from Amersham Biosciences). The purified proteins were placed in a solution of 20 mm HEPES, pH 7.5, 1 mm dithiothreitol, and 1 mm EDTA (protein concentration, 20 mg/ml). We obtained the binary complex of Dab2-DH with the 9-mer peptide of APP (–7NGYENPTYK+1) by the vapor diffusion hanging-drop method. The crystallization conditions were 3.8 m sodium formate, 50 mm HEPES, pH 8.5, at 18 °C; crystals appeared after 3–5 days. To obtain a mercury derivative, the peptide-bound crystals were soaked in a solution containing 10 mm thimerosal for 3 days. We also obtained ligand-free Dab2-DH by using a crystallization condition slightly different from that used for the binary complex (3.5 m sodium formate, 50 mm HEPES, pH 8.5 at 18 °C). We could not obtain the crystals of the ternary complex of Dab2-DH with the APP peptide and Ins-1,4,5-P3 using either the crystallization conditions of the binary complex or the ligand-free Dab2-DH. A search for new crystallization conditions for the Dab2-DH ternary complex also failed. Instead, the ternary complex crystals of Dab1-DH with the same ligands were obtained by using different crystallization conditions containing 20% (w/v) polyethylene glycol 3350, 0.2 m tri-lithium citrate at 18 °C; crystals appeared after 1 month. For data collection, we transferred the crystals to a cryoprotectant solution containing 50% mineral oil and 50% of Paraton-N light hydrocarbon oil. Native and derivative data of the binary complex of Dab2-DH were measured at 100 K on dual image plate detectors (Nonius) at St. Jude, and native data of ligand-free Dab2-DH and the ternary complex of Dab1-DH were collected at Southeast Regional Collaborative Access Team 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Crystals of the binary complex and ligand-free Dab2-DH belonged to space group R32 with unit cell dimensions of a = 127.3 Å, c = 269.5 Å and a = 128.2 Å, c = 272.3 Å. The ternary complex crystals of Dab1-DH belonged to space group P212121 with unit cell dimensions of a = 59.1 Å, b = 66.5 Å, c = 90.5 Å. Data reduction, merging, and scaling were done with the DENZO and SCALEPACK programs (41Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38250) Google Scholar). We used the single isomorphous replacement method with one heavy atom derivative (thimerosal) to determine the protein phases of the binary complex of Dab2-DH with the APP peptide. A difference Patterson map calculation, heavy atom search, electron density calculation, and density modification were done in the program CNS (42Brunger 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 (16917) Google Scholar). An atomic model was built in the program O (43Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar) and refined in the program CNS (42Brunger 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 (16917) Google Scholar). The ligand-free Dab2-DH structure was determined by the difference Fourier method because its space group and unit cell dimensions were similar to those of the binary complex of Dab2-DH. The ternary complex structure of Dab1-DH was determined by the molecular replacement method using one molecule of the binary complex structure of Dab2-DH as a search model. The cross-rotation and translation functions were calculated in the program EPMR (44Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (688) Google Scholar). Model building in the program O (43Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar) and refinement in the program CNS (42Brunger 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 (16917) Google Scholar) were iterated several times. The data collection and refinement statistics are summarized in Table I.Table IData collection and refinement statisticsThe binary complex of Dab2-DHThimerosal derivative of Dab2-DHLigand-free Dab2-DHThe ternary complex of Dab1-DHData collectionResolution (Å)20-2.4520-2.4530-2.120-2.3ReflectionTotal712,193385,1302,717,217400,319Unique31,08931,20549,67216,118R sym (%)aR sym = Σhkl[Σi|I hkl,i - 〈Ihkl〉|]/Σhkl,i 〈Ihkl〉|, where I hkl,i is the intensity of an individual measurement of the reflection with Miller indices h and l and 〈Ihkl〉 is the mean intensity of that reflection.10.09.78.47.7CompletenessTotal (%)98.799.697.199.5Last shell (%)88.2 (2.5-2.45 Å)88.3 (2.14-2.1 Å)99.4 (2.34-2.3 Å)Redundancy22.912.354.724.8Average I/σI21.617.213.527.3Phasing statisticsMean figure of merit0.29Phasing powerbPhasing power is the ratio of the root mean square value of the calculated heavy atom structure factor to the root mean square value of the difference between calculated and observed derivative structure factors, where it is averaged not only over all reflections but over all phases for each reflection, weighted by the phase probability. Centric reflections1.37 Acentric reflections1.26RefinementResolution (Å)20.0-2.4530-2.120-2.3R work (%)cR work = Σ∥F obs| - |F calc|/Σ|F obs|, where |F obs| and |F calc| are observed and calculated structure factor amplitudes, respectively.24.422.324.6R free (%)dR free is equivalent to R work except that 5% of the total reflections were set aside for an unbiased test of the progress of refinement.27.224.630.3Number of reflections28,091 (F > 0σ (F))39,909 (F > 1.5σ (F))13,125 (F > 2σ (F))Number of refined atoms3,9893,8032,803Stereochemistryr.m.s.d. bond length (Å)0.0070.0080.009r.m.s.d. bond angles (°)1.211.311.26Average B factors (Å2)42.839.247.9Residues from Ramachandran plotMost favored (%)92.694.890.0Additional allowed (%)7.45.210.0a R sym = Σhkl[Σi|I hkl,i - 〈Ihkl〉|]/Σhkl,i 〈Ihkl〉|, where I hkl,i is the intensity of an individual measurement of the reflection with Miller indices h and l and 〈Ihkl〉 is the mean intensity of that reflection.b Phasing power is the ratio of the root mean square value of the calculated heavy atom structure factor to the root mean square value of the difference between calculated and observed derivative structure factors, where it is averaged not only over all reflections but over all phases for each reflection, weighted by the phase probability.c R work = Σ∥F obs| - |F calc|/Σ|F obs|, where |F obs| and |F calc| are observed and calculated structure factor amplitudes, respectively.d R free is equivalent to R work except that 5% of the total reflections were set aside for an unbiased test of the progress of refinement. Open table in a new tab Nuclear Magnetic Resonance—The NMR samples contained 1 mm uniformly 15N-labeled sample (wild-type Dab1-DH or one of its two mutants, L45A and K82A, in a solution of 40 mm phosphate, 6 mm deuterated dithiothreitol, pH 6.8, and 5% D2O. Ins-1,4,5-P3 (Sigma) in the same solution was titrated into the protein sample during the course of the NMR experiments. Two-dimensional 15N heteronuclear single quantum coherence spectroscopy (HSQC) spectra were measured with a Varian INOVA 600-MHz NMR spectrometer at 25 °C. Chemical shift perturbation experiments were performed by measuring the chemical shift differences in the HSQC spectra as wild-type Dab1-DH was titrated with Ins-1,4,5-P3. In addition, the HSQC chemical shifts of the two mutants were measured and compared with that of the wild-type measured before Ins-1,4,5-P3 titration. Data were processed and displayed by the program packages NMRpipe and NMRDraw (45Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11278) Google Scholar). The resulting spectra were analyzed using SPARKY (46Goddard T.D. Kneller D.G. SPARKY. University of California at San Francisco, San Francisco, CA2002Google Scholar). Mutagenesis and Peptide Binding Assay—The construction of a Dab1-hemagglutinin expression plasmid has been described previously (47Keshvara L. Benhayon D. Magdaleno S. Curran T. J. Biol. Chem. 2001; 276: 16008-16014Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Site-directed mutagenesis was conducted using the QuikChange site-directed mutagenesis kit (Stratagene). A synthetic peptide corresponding to the NPXY-containing cytoplasmic domain of amyloid precursor-like protein 1 (–12CELQRHGYENPTYRFLEE+5) and a random peptide used as a negative control (CFEYRNRHQETPELLGEY) have been described previously (18Homayouni R. Rice D.S. Sheldon M. Curran T. J. Neurosci. 1999; 19: 7507-7515Crossref PubMed Google Scholar). The peptides were coupled to Sepharose by using the SulfoLink Immobilization kit (Pierce). HEK293T cells were transiently transfected with expression plasmids for either wild-type Dab1 or mutant Dab1 carrying single amino acid substitutions (S114T, H136R, and F158V). After 24 h, cells were lysed in cell lysis buffer (25 mm Tris-HCl, 1% Nonidet P-40, 150 mm NaCl, 5 mm EDTA, 1 mm sodium orthovanadate, 20 mm sodium fluoride, and 20 μg/ml each of aprotinin and leupeptin). The lysates were incubated with either the amyloid precursor-like protein 1 NPXY peptide or the control peptide at 4 °C for 2 h. The peptide precipitates were washed three times with the cell lysis buffer, and the proteins were separated by SDS-PAGE. After transfer to nitrocellulose membrane, bound Dab1 was detected by Western blotting with anti-Dab1 antibodies. To confirm the expression of Dab1 and Dab1 mutants, 10% of the lysate input was analyzed on a separate gel. Super Signal West Dura Extended Duration Substrate (Pierce) was used for detection. Mutagenesis and PtdIns-P2 Binding Assay—DNA fragments encoding Dab1-DH and Dab2-DH were cloned into pET28a vector between the NdeI and SalI sites. Single and double mutations at the proposed phosphoinositide-binding site were introduced by using the QuikChange site-directed mutagenesis kit (Stratagene). The mutations were K45A and K85A of Dab1-DH and K53A and K90A of Dab2-DH. All clones and mutations were checked by automated sequencing on the ABI Prism 3700 DNA Analyzer. His-tagged wild-type and mutant proteins were expressed in BL21(DE3) cells and separated in nickel nitrilotriacetic acid-agarose gel (Qiagen, Valencia, CA). The proper folding of the mutant proteins was confirmed by circular dichroism spectroscopy. The PtdIns-4,5-P2 binding ability of Dab1-DH and Dab2-DH were assessed by protein-lipid binding assay with the His-tagged fusion proteins and PtdIns-4,5-P2 membrane strips (Echelon Biosciences Inc.). The membrane spotted with PtdIns-4,5-P2 was incubated with TBS-T (20 mm Tris-HCl, pH 7.6, 150 mm NaCl, and 0.1% (v/v) Triton X-100) containing 3% fatty acid-free BSA for 1 h at room temperature to block nonspecific reactions and was then incubated overnight at 4 °C with 500 ng/ml indicated His-tagged protein in TBS-T with 3% fatty acid-free BSA with gentle shaking. The membrane was washed 3 times over 30 min in TBS-T containing 3% BSA and then incubated for 1 h with a 1:500 dilution of rabbit anti-His polyclonal IgG (Santa Cruz Biotechnology, CA) in TBS-T with 1% fatty acid-free BSA. The membrane was washed 4 times over 1 h in TBS-T and then incubated for 2 h with a 1:5000 dilution of anti-rabbit polyclonal IgG linked to alkaline phosphatase in TBS-T with 1% fatty acid-free BSA. Finally, the membrane was washed 4 times over 1 h in TBS" @default.
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• W2022742531 title "Crystal Structures of the Dab Homology Domains of Mouse Disabled 1 and 2" @default.
• W2022742531 cites W1508994506 @default.
• W2022742531 cites W1539796472 @default.
• W2022742531 cites W1541258411 @default.
• W2022742531 cites W1562466038 @default.
• W2022742531 cites W1821101374 @default.
• W2022742531 cites W1865164120 @default.
• W2022742531 cites W1966161684 @default.
• W2022742531 cites W1971587010 @default.
• W2022742531 cites W1977170693 @default.
• W2022742531 cites W1977412572 @default.
• W2022742531 cites W1982750125 @default.
• W2022742531 cites W1983795337 @default.
• W2022742531 cites W1983921682 @default.
• W2022742531 cites W1991203655 @default.
• W2022742531 cites W1994063020 @default.
• W2022742531 cites W1995017064 @default.
• W2022742531 cites W1997140657 @default.
• W2022742531 cites W2002390599 @default.
• W2022742531 cites W2002760965 @default.
• W2022742531 cites W2005841227 @default.
• W2022742531 cites W2008754491 @default.
• W2022742531 cites W2011073796 @default.
• W2022742531 cites W2013083986 @default.
• W2022742531 cites W2020952581 @default.
• W2022742531 cites W2029675956 @default.
• W2022742531 cites W2032062053 @default.
• W2022742531 cites W2033822739 @default.
• W2022742531 cites W2036088572 @default.
• W2022742531 cites W2038403184 @default.
• W2022742531 cites W2039836490 @default.
• W2022742531 cites W2039895922 @default.
• W2022742531 cites W2042674639 @default.
• W2022742531 cites W2044965047 @default.
• W2022742531 cites W2050193310 @default.
• W2022742531 cites W2051758765 @default.
• W2022742531 cites W2056281513 @default.
• W2022742531 cites W2060650825 @default.
• W2022742531 cites W2068963464 @default.
• W2022742531 cites W2069116251 @default.
• W2022742531 cites W2070362107 @default.
• W2022742531 cites W2072021181 @default.
• W2022742531 cites W2079211667 @default.
• W2022742531 cites W2079252365 @default.
• W2022742531 cites W2088882706 @default.
• W2022742531 cites W2089828884 @default.
• W2022742531 cites W2092249847 @default.
• W2022742531 cites W2098494612 @default.
• W2022742531 cites W2099022660 @default.
• W2022742531 cites W2113438288 @default.
• W2022742531 cites W2118059649 @default.
• W2022742531 cites W2119766505 @default.
• W2022742531 cites W2120982408 @default.
• W2022742531 cites W2139474686 @default.
• W2022742531 cites W2145295859 @default.
• W2022742531 cites W2147911445 @default.
• W2022742531 cites W2162163372 @default.
• W2022742531 cites W2162321314 @default.
• W2022742531 cites W2168721537 @default.
• W2022742531 cites W2169821755 @default.
• W2022742531 cites W32449160 @default.
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