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Structure of the C-terminal Phosphotyrosine Interaction Domain of Fe65L1 Complexed with the Cytoplasmic Tail of Amyloid Precursor Protein Reveals a Novel Peptide Binding Mode

2008; Elsevier BV; Volume: 283; Issue: 40 Linguagem: Inglês

10.1074/jbc.m803892200

ISSN

1083-351X

Autores

Hua Li, S. Koshiba, Fumiaki Hayashi, N. Tochio, T. Tomizawa, T. Kasai, Takashi Yabuki, Yoko Motoda, T. Harada, Satoru Watanabe, Makoto Inoue, Yoshihide Hayashizaki, Akiko Tanaka, T. Kigawa, Shigeyuki Yokoyama,

Tópico(s)

Enzyme Structure and Function

Resumo

Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I β-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel β-sheet with the β5 strand of the PID domain, the binding mode typically observed in the PID/PTB·peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn α-helix and interacts extensively with the C-terminal α-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB·peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins. Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I β-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel β-sheet with the β5 strand of the PID domain, the binding mode typically observed in the PID/PTB·peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn α-helix and interacts extensively with the C-terminal α-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB·peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins. Alzheimer disease is a neurodegenerative disorder characterized by senile plaques and neurofibrillary tangles. The predominant constituent of the amyloid is the 39–43-residue amyloid β peptide (Aβ), 3The abbreviations used are: Aβ, amyloid β peptide; APP, amyloid precursor protein; PID/PTB, phosphotyrosine interaction/phosphotyrosine binding; Dab, Disabled; JIP, c-Jun N-terminal protein kinase-interacting protein; ITC, isothermal titration calorimetry; DTT, dithiothreitol; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; NOE, nuclear Overhauser effect. 3The abbreviations used are: Aβ, amyloid β peptide; APP, amyloid precursor protein; PID/PTB, phosphotyrosine interaction/phosphotyrosine binding; Dab, Disabled; JIP, c-Jun N-terminal protein kinase-interacting protein; ITC, isothermal titration calorimetry; DTT, dithiothreitol; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; NOE, nuclear Overhauser effect. a proteolytic cleavage product of the amyloid precursor protein (APP) (1.Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5121) Google Scholar). APP is an integral transmembrane glycoprotein composed of a large extracellular domain, a single membrane-spanning region, and a short cytoplasmic domain (see Fig. 1A). The APP gene encodes several different isoforms of APP as a consequence of alternative splicing. The three major isoforms are APP770, APP751, and APP695, which all possess the same 47-residue cytoplasmic domain. The functional role of APP remains unclear; however, several lines of evidence suggest that APP is part of a diverse protein-protein interaction network, which is centered on the short cytoplasmic domain. The cytoplasmic domain participates in the important cellular processes of intracellular trafficking and secretion of APP and signal transduction via interactions with adaptor and signaling proteins, respectively. Several proteins reportedly interact with the cytoplasmic domain of APP. These include heterotrimeric G protein Go (2.Nishimoto I. Okamoto T. Matsuura Y. Takahashi S. Okamoto T. Murayama Y. Ogata E. Nature. 1993; 362: 75-79Crossref PubMed Scopus (370) Google Scholar), the 59-kDa ubiquitously expressed protein APP-BP1 (3.Chow N. Korenberg J.R. Chen X.N. Neve R.L. J. Biol. Chem. 1996; 271: 11339-11346Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), the neuron-specific X11 protein (4.Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (430) Google Scholar), Fe65 family proteins (4.Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (430) Google Scholar, 5.Fiore F. Zambrano N. Minopoli G. Donini V. Duilio A. Russo T. J. Biol. Chem. 1995; 270: 30853-30856Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 6.McLoughlin D.M. Miller C.C. FEBS Lett. 1996; 397: 197-200Crossref PubMed Scopus (133) Google Scholar, 7.Guénette S.Y. Chen J. Jondro P.D. Tanzi R.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10832-10837Crossref PubMed Scopus (149) Google Scholar, 8.Zambrano N. Buxbaum J.D. Minopoli G. Fiore F. De Candia P. De Renzis S. Faraonio R. Sabo S. Cheetham J. Sudol M. Russo T. J. Biol. Chem. 1997; 272: 6399-6405Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 9.Duilio A. Faraonio R. Minopoli G. Zambrano N. Russo T. Biochem. J. 1998; 330: 513-519Crossref PubMed Scopus (86) Google Scholar), mammalian Disabled (Dab) protein (10.Homayouni R. Rice D.S. Sheldon M. Curran T. J. Neurosci. 1999; 19: 7507-7515Crossref PubMed Google Scholar, 11.Howell B.W. Lanier L.M. Frank R. Gertler F.B. Cooper J.A. Mol. Cell. Biol. 1999; 19: 5179-5188Crossref PubMed Scopus (332) Google Scholar), and c-Jun N-terminal protein kinase-interacting protein (JIP) (12.Inomata H. Nakamura Y. Hayakawa A. Takata H. Suzuki T. Miyazawa K. Kitamura N. J. Biol. Chem. 2003; 278: 22946-22955Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 13.Scheinfeld M.H. Roncarati R. Vito P. Lopez P.A. Abdallah M. D'Adamio L. J. Biol. Chem. 2002; 277: 3767-3775Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) as well as the microtubule-binding protein PAT1 (14.Zheng P. Eastman J. Vande Pol S. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14745-14750Crossref PubMed Scopus (107) Google Scholar). Some of these proteins are phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain-containing proteins, including X11 (4.Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (430) Google Scholar), Fe65 (4.Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (430) Google Scholar, 5.Fiore F. Zambrano N. Minopoli G. Donini V. Duilio A. Russo T. J. Biol. Chem. 1995; 270: 30853-30856Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 6.McLoughlin D.M. Miller C.C. FEBS Lett. 1996; 397: 197-200Crossref PubMed Scopus (133) Google Scholar, 7.Guénette S.Y. Chen J. Jondro P.D. Tanzi R.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10832-10837Crossref PubMed Scopus (149) Google Scholar, 8.Zambrano N. Buxbaum J.D. Minopoli G. Fiore F. De Candia P. De Renzis S. Faraonio R. Sabo S. Cheetham J. Sudol M. Russo T. J. Biol. Chem. 1997; 272: 6399-6405Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 9.Duilio A. Faraonio R. Minopoli G. Zambrano N. Russo T. Biochem. J. 1998; 330: 513-519Crossref PubMed Scopus (86) Google Scholar), Dab (10.Homayouni R. Rice D.S. Sheldon M. Curran T. J. Neurosci. 1999; 19: 7507-7515Crossref PubMed Google Scholar, 11.Howell B.W. Lanier L.M. Frank R. Gertler F.B. Cooper J.A. Mol. Cell. Biol. 1999; 19: 5179-5188Crossref PubMed Scopus (332) Google Scholar), and JIP (12.Inomata H. Nakamura Y. Hayakawa A. Takata H. Suzuki T. Miyazawa K. Kitamura N. J. Biol. Chem. 2003; 278: 22946-22955Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 13.Scheinfeld M.H. Roncarati R. Vito P. Lopez P.A. Abdallah M. D'Adamio L. J. Biol. Chem. 2002; 277: 3767-3775Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). They recognize the NPTY sequence within the cytoplasmic domain of APP.The Fe65 family proteins, Fe65, Fe65L1, and Fe65L2, are adaptor proteins that possess three protein-protein interaction domains: one WW domain and two PID/PTB domains (see Fig. 1B) (15.Russo T. Faraonio R. Minopoli G. De Candia P. De Renzis S. Zambrano N. FEBS Lett. 1998; 434: 1-7Crossref PubMed Scopus (97) Google Scholar). The N- and C-terminal PID/PTB domains are referred to as PID1 and PID2, respectively. The three protein-protein interaction domains are well conserved among the Fe65 family proteins, sharing 50–60% amino acid sequence identity, whereas most of the remaining parts of the proteins are unrelated. Among the three Fe65 family proteins, the most significant difference is their tissue distribution: Fe65 mRNA is neuron-specific (16.Duilio A. Zambrano N. Mogavero A.R. Ammendola R. Cimino F. Russo T. Nucleic Acids Res. 1991; 19: 5269-5274Crossref PubMed Scopus (87) Google Scholar), whereas Fe65L1 mRNA is ubiquitously expressed (7.Guénette S.Y. Chen J. Jondro P.D. Tanzi R.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10832-10837Crossref PubMed Scopus (149) Google Scholar), and Fe65L2 mRNA significantly accumulates in the brain and testis (9.Duilio A. Faraonio R. Minopoli G. Zambrano N. Russo T. Biochem. J. 1998; 330: 513-519Crossref PubMed Scopus (86) Google Scholar). The Fe65 gene was originally isolated as a neuron-specific gene, and it has some characteristics of a transcription factor (16.Duilio A. Zambrano N. Mogavero A.R. Ammendola R. Cimino F. Russo T. Nucleic Acids Res. 1991; 19: 5269-5274Crossref PubMed Scopus (87) Google Scholar, 17.Simeone A. Duilio A. Fiore F. Acampora D. De Felice C. Faraonio R. Paolocci F. Cimino F. Russo T. Dev. Neurosci. 1994; 16: 53-60Crossref PubMed Scopus (30) Google Scholar). The interaction between the Fe65 family proteins and the APP cytoplasmic domain has been confirmed both in vitro (5.Fiore F. Zambrano N. Minopoli G. Donini V. Duilio A. Russo T. J. Biol. Chem. 1995; 270: 30853-30856Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 7.Guénette S.Y. Chen J. Jondro P.D. Tanzi R.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10832-10837Crossref PubMed Scopus (149) Google Scholar) and in vivo (8.Zambrano N. Buxbaum J.D. Minopoli G. Fiore F. De Candia P. De Renzis S. Faraonio R. Sabo S. Cheetham J. Sudol M. Russo T. J. Biol. Chem. 1997; 272: 6399-6405Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). For all three Fe65 family proteins, the C-terminal PID/PTB domain (PID2) was demonstrated to be sufficient for their binding to the cytoplasmic domain of APP (4.Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (430) Google Scholar). Furthermore phosphorylation of the tyrosine in the NPTY motif is not required (4.Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (430) Google Scholar). On the other hand, a yeast two-hybrid screening study (5.Fiore F. Zambrano N. Minopoli G. Donini V. Duilio A. Russo T. J. Biol. Chem. 1995; 270: 30853-30856Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) and a peptide competition experiment (8.Zambrano N. Buxbaum J.D. Minopoli G. Fiore F. De Candia P. De Renzis S. Faraonio R. Sabo S. Cheetham J. Sudol M. Russo T. J. Biol. Chem. 1997; 272: 6399-6405Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) showed that a 32-residue-long peptide, DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN, located at the extreme C terminus of the cytoplasmic domain of APP, was necessary for binding PID2 of Fe65. The 32-residue-long peptide (termed APP-32mer in Fig. 1A), including the NPTY motif, is much longer than the peptide that was reported previously to be recognized by the PID/PTB domain. The YENPTY sequence is also a sorting motif, or an internalization motif, required for trafficking of APP into the endocytic pathway (18.Perez R.G. Soriano S. Hayes J.D. Ostaszewski B. Xia W. Selkoe D.J. Chen X. Stokin G.B. Koo E.H. J. Biol. Chem. 1999; 274: 18851-18856Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Studies have revealed that Fe65 family proteins can alter the processing of APP by influencing APP trafficking (19.Sabo S.L. Lanier L.M. Ikin A.F. Khorkova O. Sahasrabudhe S. Greengard P. Buxbaum J.D. J. Biol. Chem. 1999; 274: 7952-7957Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 20.Guénette S.Y. Chen J. Ferland A. Haass C. Capell A. Tanzi R.E. J. Neurochem. 1999; 73: 985-993Crossref PubMed Scopus (72) Google Scholar, 21.Chang Y. Tesco G. Jeong W.J. Lindsley L. Eckman E.A. Eckman C.B. Tanzi R.E. Guénette S.Y. J. Biol. Chem. 2003; 278: 51100-51170Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Fe65 has been shown to increase α-secretase-cleaved APP and Aβ production (19.Sabo S.L. Lanier L.M. Ikin A.F. Khorkova O. Sahasrabudhe S. Greengard P. Buxbaum J.D. J. Biol. Chem. 1999; 274: 7952-7957Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Fe65L1 also promotes α-secretase-cleaved APP secretion and APP maturation (20.Guénette S.Y. Chen J. Ferland A. Haass C. Capell A. Tanzi R.E. J. Neurochem. 1999; 73: 985-993Crossref PubMed Scopus (72) Google Scholar). These regulatory activities of Fe65L1 require the binding of Fe65L1 to APP C-terminal fragments (21.Chang Y. Tesco G. Jeong W.J. Lindsley L. Eckman E.A. Eckman C.B. Tanzi R.E. Guénette S.Y. J. Biol. Chem. 2003; 278: 51100-51170Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).Although the interaction between APP and the Fe65 family proteins is highly significant, in terms of the biology of Alzheimer disease, little is known about the molecular basis of the interaction. Here we report the solution structure of PID2 of mouse Fe65L1 in the free form and in complex with APP-32mer and the characterization of their interaction by isothermal titration calorimetry (ITC) and NMR spectroscopy. Among the three Fe65 family proteins, we selected PID2 of Fe65L1 as a target because the mRNA encoding Fe65L1 is ubiquitously expressed. To facilitate the structure determination of the complex of PID2 of Fe65L1 and APP-32mer, we designed several different chimeric proteins in which the corresponding regions of PID2 and APP-32mer were integrated. The complex structure reported here reveals a novel peptide binding mode as compared with those of the canonical PID domains that recognize the NPX(p)Y motif ((p) indicates that tyrosine may be phosphorylated).EXPERIMENTAL PROCEDURESExpression and Purification of PID2 of Fe65L1—The DNA fragment encoding PID2 of mouse Fe65L1 (amino acid residues Pro582 to Cys704; Swiss-Prot accession number Q9DBR4) was amplified via PCR from the RIKEN full-length enriched mouse cDNA library (Clone ID 1200015I07) (22.RIKEN Genome Exploration Research Group Phase II Team and FANTOM ConsortiumNature. 2001; 409: 685-690Crossref PubMed Scopus (570) Google Scholar, 23.FANTOM Consortium and RIKEN Genome Exploration Research Group Phase I & II TeamNature. 2002; 420: 563-573Crossref PubMed Scopus (1373) Google Scholar) and was cloned into the plasmid vector pCR2.1 (Invitrogen) as a fusion with an N-terminal His tag and a tobacco etch virus protease cleavage site. The 13C,15N-labeled protein was synthesized by the cell-free protein expression system (24.Kigawa T. Yabuki T. Yoshida Y. Tsutsui M. Ito Y. Shibata T. Yokoyama S. FEBS Lett. 1999; 442: 15-19Crossref PubMed Scopus (424) Google Scholar, 25.Kigawa T. Yabuki T. Matsuda N. Matsuda T. Nakajima R. Tanaka A. Yokoyama S. J. Struct. Funct. Genomics. 2004; 5: 63-68Crossref PubMed Scopus (268) Google Scholar, 26.Matsuda T. Koshiba S. Tochio N. Seki E. Iwasaki N. Yabuki T. Inoue M. Yokoyama S. Kigawa T. J. Biomol. NMR. 2007; 37: 225-229Crossref PubMed Scopus (62) Google Scholar). The cell-free reaction solution was first absorbed to a TALON affinity column, which was washed with 20 mm Tris-HCl buffer (pH 8.0) containing 1 m NaCl, and was eluted with 20 mm Tris-HCl buffer (pH 8.0) containing 300 mm NaCl and 500 mm imidazole. The solution was then desalted on a HiPrep 26/10 Desalting column with 20 mm Tris-HCl buffer (pH 8.0) containing 300 mm NaCl. The His tag was cleaved by an incubation at 30 °C for 1 h with tobacco etch virus protease. The sample was then loaded on a TALON affinity column, which was washed with 20 mm Tris-HCl buffer (pH 8.0) containing 300 mm NaCl, and was eluted with 20 mm Tris-HCl buffer (pH 8.0) containing 300 mm NaCl and 500 mm imidazole. The flow-through fraction was desalted on a HiPrep 26/10 Desalting column with 20 mm Tris-HCl buffer (pH 8.0) containing 1 mm EDTA. The sample was then loaded on a HiTrap Q column, which was washed with 20 mm Tris-HCl buffer (pH 8.0) containing 1 mm EDTA, and was eluted with a concentration gradient of 20 mm Tris-HCl buffer (pH 8.0) containing 1 mm EDTA and 1 m NaCl. Finally the sample was purified on a HiLoad 16/60 Superdex75 column, which was washed with 20 mm Tris-HCl buffer (pH 7.0) containing 200 mm NaCl, 1 mm EDTA, and 1 mm DTT. The fraction containing the PID domain was concentrated to 1.49 mg/ml in 20 mm Tris-HCl buffer (pH 7.0) containing 200 mm NaCl, 1 mm EDTA, 1 mm DTT, and protease inhibitor mixture (Complete, EDTA-free (Roche Applied Science)). For the NMR structure determination, a 0.84 mm sample of free PID2 was prepared in 20 mm deuterated Tris buffer (pH 7.0) containing 100 mm NaCl, 2 mm deuterated DTT, 0.02% NaN3, and 10% 2H2O, 90% 1H2O. The protein sample for the NMR measurements consisted of 136 amino acid residues. The first 7 amino acid residues at the N terminus (GSSGSSG) and the last 6 residues at the C terminus (SGPSSG) were derived from the linker sequence used in the expression and purification system.NMR Sample Preparation of the PID2 and APP-32mer Complex—To prepare the sample of the PID2 and APP-32mer complex for NMR measurement, a solution of non-labeled APP-32mer (Toray Research Center, Tokyo, Japan), dissolved in buffer A (20 mm deuterated Tris buffer (pH 7.0) containing 100 mm NaCl, 1 mm deuterated DTT, 0.02% NaN3, and 10% 2H2O, 90% 1H2O) with pH adjustment, was gradually added to 13C,15N-labeled PID2, which had been buffer-exchanged in buffer A, until the free form of PID2 was completely converted into the peptide-bound form as confirmed by the 1H-15N HSQC spectra. The final concentration of PID2 was 0.37 mm.Design and Expression of the PID2-APP-32mer Chimera—To prepare the PID2-APP-32mer chimera, the gene encoding PID2 was fused to the cDNA of APP-32mer (amino acid residues Asp739*4 to Asn770*; Swiss-Prot accession number P12023), and the resulting gene was inserted within the plasmid vector pCR2.1 (Invitrogen), incorporating an N-terminal His tag and a tobacco etch virus cleavage site. The 13C,15N-labeled chimera was synthesized by the cell-free protein expression system (24.Kigawa T. Yabuki T. Yoshida Y. Tsutsui M. Ito Y. Shibata T. Yokoyama S. FEBS Lett. 1999; 442: 15-19Crossref PubMed Scopus (424) Google Scholar, 25.Kigawa T. Yabuki T. Matsuda N. Matsuda T. Nakajima R. Tanaka A. Yokoyama S. J. Struct. Funct. Genomics. 2004; 5: 63-68Crossref PubMed Scopus (268) Google Scholar, 26.Matsuda T. Koshiba S. Tochio N. Seki E. Iwasaki N. Yabuki T. Inoue M. Yokoyama S. Kigawa T. J. Biomol. NMR. 2007; 37: 225-229Crossref PubMed Scopus (62) Google Scholar). Three kinds of chimeras, differing in the relative positions of PID2 and APP-32mer in the protein, were designed for the structure determination (Fig. 1C). The three chimeras were designated as I, II, and III, respectively. The construct of Chimera I included, from the N to the C terminus, a His tag, APP-32mer, a 14-residue linker (SGSSGSSGSSGSSG), and PID2. The Chimera II construct consisted of a His tag, APP-32mer, a 23-residue linker containing a protease Factor Xa cleavage site (SGPSSGIEGRGSSGSSGSSGSSG), and PID2; the construct of Chimera III included a His tag, PID2, the 23-residue linker, and APP-32mer. The 13C,15N-labeled chimeras were synthesized and purified as described below. After the His tag cleavage in the purification procedure, Chimera I consisted of 176 amino acid residues; both Chimeras II and III consisted of 185 amino acid residues. The first 7 amino acid residues at the N terminus (GSSGSSG) in each sample were derived from the linker sequence used in the expression and purification system.Purification of the PID2-APP-32mer Chimeras—The cell-free reaction solution of each chimera was adsorbed onto a HiTrap chelating column (Amersham Biosciences), which was washed with 20 mm Tris-HCl buffer (pH 8.0) containing 1 m NaCl and 20 mm imidazole, and was eluted with 20 mm Tris-HCl buffer (pH 8.0) containing 500 mm NaCl and 500 mm imidazole. To cleave the His tag, the eluted sample was incubated with tobacco etch virus protease at 30 °C for 3 h for Chimeras I and II and overnight for Chimera III. The sample was then loaded on a HiPrep 26/10 Desalting column, which was eluted with 20 mm Tris-HCl buffer (pH 8.0) containing 300 mm NaCl and 20 mm imidazole. The fractionated dialysate was applied to a HiTrap chelating column, which was equilibrated with 20 mm Tris-HCl buffer (pH 8.0) containing 300 mm NaCl and 20 mm imidazole, and was eluted with a concentration gradient of imidazole (500 mm final concentration). The eluted fraction of each chimera sample was concentrated in 20 mm Tris-HCl buffer (pH 8.0) containing 300 mm NaCl, 1 mm DTT, 0.5 mm EDTA, and protease inhibitor mixture (Complete Mini). Each NMR sample contained ∼1.0 mm of uniformly 13C,15N-labeled chimera in 20 mm deuterated Tris buffer (pH 7.0) containing 100 mm NaCl, 1 mm deuterated DTT, 0.02% NaN3, and 10% 2H2O, 90% 1H2O.ITC Measurements—ITC measurements were performed on a MicroCal VP-ITC isothermal titration calorimeter (Micro-Cal, Inc.). HEPES buffer (20 mm HEPES buffer (pH 7.0) containing 100 mm NaCl and 1 mm DTT) was used for the ITC measurements. PID2 was buffer-exchanged in the ITC buffer, and APP-32mer was dissolved in the same buffer with pH adjustment. The concentrations of PID2 and APP-32mer used for the ITC measurement were 0.109 and 1.640 mm, respectively, as determined using UV absorbance values measured at 280 nm. A degassed sample of PID2 was kept at room temperature (25 °C) and was stirred at 307 rpm in a 1.4-ml reaction cell. For each titration, 5-μl aliquots of peptide were delivered into the PID2 solution at 240-s intervals to allow complete equilibration. The final ratio of peptide to protein reached 2:1 at the end of the titration. Heat transfer was measured as a function of elapsed time. The heat of dilution, obtained by titrating the identical peptide solution into the reaction cell containing only the HEPES buffer, was subtracted prior to analysis. The corrected titration curve was fitted with a one-site model, and the thermodynamic parameters were calculated using the Origin software (version 7.0) provided by MicroCal.Titration Experiments by NMR—NMR titration experiments were carried out by adding the unlabeled APP-32mer solution to 13C,15N-labeled PID2. In total, four NMR samples were prepared for the titration experiment. In the four NMR samples, the concentration of PID2 was maintained at 0.050 mm, and the concentration of APP-32mer was varied to generate a series of different PID2:APP-32mer molar ratios (1:0, 1:0.5, 1:1, and 1:1.5). The buffer used for the APP-32mer titration was the same as that used for the structure determination. 1H-15N HSQC spectra were measured after each titration step, and the signals were monitored by observing the changes in the chemical shifts of the amide signals in 1H-15N HSQC spectra. The weighted chemical shift change (in ppm units) of the amide proton (ΔδHN) and nitrogen (ΔδN) was calculated according to the following equation: Δδtotal = [(ΔδHNWHN)2 + (ΔδNWN)2]½ where WHN = 1 and WN = 0.154 (27.Ayed A. Mudler F.A. Yi G.S. Lu Y. Kay L.E. Arrowsmith C.H. Nat. Struct. Biol. 2001; 8: 756-760Crossref PubMed Scopus (230) Google Scholar).NMR Spectroscopy—The NMR data for PID2 in the free form were recorded at 298 K on Varian Inova 600- and 900-MHz spectrometers equipped with pulsed field gradient probes. The NMR data for the three chimeras, Chimera I, Chimera II, and Chimera III, and PID2 complexed with non-labeled APP-32mer were recorded at 296 K on a Bruker AVANCE 700-MHz spectrometer, which was equipped with a triple resonance cryoprobe, and on AVANCE 800- and 900-MHz spectrometers. Sequence-specific resonance assignments were made using the standard triple resonance techniques (28.Bax A. Curr. Opin. Struct. Biol. 1994; 4: 738-744Crossref Scopus (191) Google Scholar). For PID2 complexed with the non-labeled APP-32mer, two-dimensional [F2] 13C,15N-filtered total correlation spectroscopy and two-dimensional [F1,F2] 13C,15N-filtered NOESY (29.Iwahara J. Wojciak J.M. Clubb R.T. J. Biomol. NMR. 2001; 19: 231-241Crossref PubMed Scopus (55) Google Scholar) were measured for the chemical shift assignments of the non-labeled APP-32mer, and two-dimensional [F2] 13C,15N-filtered NOESY (29.Iwahara J. Wojciak J.M. Clubb R.T. J. Biomol. NMR. 2001; 19: 231-241Crossref PubMed Scopus (55) Google Scholar), three-dimensional 13C,15N F1-filtered, 13C F3-edited NOESY and three-dimensional 13C,15N F1-filtered, 15N F3-edited NOESY (30.Zwahlen C. Legault P. Vincent S.J.F. Greenblatt J. Konrat R. Kay L.E. J. Am. Chem. Soc. 1997; 119: 6711-6721Crossref Scopus (536) Google Scholar) with mixing times of 80, 100, and 100 ms, respectively, were recorded for the detection of intermolecular NOEs. The filter-related spectra were measured on an 800-MHz spectrometer. All of the spectra were processed using the NMRPipe software package (31.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11388) Google Scholar). The programs KUJIRA (32.Kobayashi N. Iwahara J. Koshiba S. Tomizawa T. Tochio N. Güntert P. Kigawa T. Yokoyama S. J. Biomol. NMR. 2007; 39: 31-52Crossref PubMed Scopus (133) Google Scholar) and NMRView (33.Johnson B. Blevins R. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2662) Google Scholar, 34.Johnson B. Methods Mol. Biol. 2004; 278: 313-352PubMed Google Scholar) were used for visualization of the NMR spectra and chemical shift assignments.Structure Calculations—For the structure calculations of PID2 in the free form, Chimera I, Chimera II, and Chimera III, 15N-edited NOESY and 13C-edited NOESY with 80-ms mixing times were used to determine the distance restraints. Dihedral angle restraints were derived using the program TALOS (35.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2729) Google Scholar). The stereospecific assignments of the Val and Leu methyl groups were determined when they were distinguishable from their NOE patterns. Automated NOE cross-peak assignments and structure calculations with torsion angle dynamics were performed using the program CYANA (36.Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2545) Google Scholar, 37.Herrmann T. Güntert P. Wüthrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1321) Google Scholar, 38.Jee J. Güntert P. J. Struct. Funct. Genomics. 2003; 4: 179-189Crossref PubMed Scopus (83) Google Scholar, 39.Güntert P. Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1151) Google Scholar).For the structure calculation of PID2 in complex with the non-labeled APP-32mer, the stereospecific assignments and the dihedral angle restraints for the PID2 part were added in a way similar to that described above. The dihedral angle restraints for APP-32mer were only added in its secondary structural region by checking the corresponding NOE patterns. In addition, a total of five NOE peak lists were used in the complex structure calculation. Among the five peak lists, two were obtained from three-dimensional 13C-edited NOESY-HSQC and 15N-edited NOESY-HSQC, and three were obtained from two-dimensional [F2] 13C,15N-filtered NOESY, three-dimensional 13C,15N F1-filtered, 13C F3-edited NOESY, and three-dimensional 13C,15N F1-filtered, 15N F3-edited NOESY. Dihedral angle restraints and stereospecific assignments of the Val and Leu methyl groups were obtained in a similar manner as described above. The peak list from two-dimensional [F2] 13C,15N-filtered NOESY provided information about the intramolecular NOEs of APP-32mer and the intermolecular NOEs between PID2 and A

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