Solution Structure of Porcine Delta Sleep-inducing Peptide Immunoreactive Peptide A Homolog of the ShortsightedGene Product
1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês
10.1074/jbc.272.49.30918
ISSN1083-351X
AutoresG. Seidel, Knut Adermann, Thomas H. Schindler, Andrzej Ejchart, Rainer Jaenicke, Wolf‐Georg Forssmann, Paul Rösch,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9–46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9–46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i′ + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed. The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9–46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9–46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i′ + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed. Delta sleep-inducing peptide immunoreactive peptide (DIP) 1The abbreviations used are: DIP, delta sleep-inducing peptide; pDIP, porcine DIP; hDIP, human DIP; bZIP, basic region/leucine zipper; MD, molecular dynamics; RMSD, root mean square deviation; Fmoc, N-(9-fluorenyl)methoxycarbonyl; TBTU,O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate; HPLC, high pressure liquid chromatography; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy. 1The abbreviations used are: DIP, delta sleep-inducing peptide; pDIP, porcine DIP; hDIP, human DIP; bZIP, basic region/leucine zipper; MD, molecular dynamics; RMSD, root mean square deviation; Fmoc, N-(9-fluorenyl)methoxycarbonyl; TBTU,O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate; HPLC, high pressure liquid chromatography; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy. is a 77-residue NH2-terminally acetylated peptide that was originally isolated from porcine brain (pDIP) using polyclonal antibodies against the delta sleep-inducing peptide (DSIP) (1Monnier M. Dudler L. Gächter R. Maier P.F. Tobler H.J. Schoenenberger G.A. Experimentia (Basel). 1977; 33: 548-552Crossref PubMed Scopus (43) Google Scholar, 2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar). Although DIP was detected via DSIP-specific antibodies that may recognize the sequence GGDA in DSIP and GGSA in pDIP, it is otherwise not sequence-related to this supposed sleep-inducing peptide. The pDIP sequence contains a putative leucine-zipper motif, a Pro/Glu rich domain, and three potential phosphorylation sites (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar). DNA-binding capability of pDIP, however, is not obvious from its sequence, because it lacks the basic region found in the original basic region/leucine zipper (bZIP) DNA-binding domains. In the bZIP family of transcription factors, the leucine zipper acts as a dimerization domain and the upstream basic region as a DNA-binding domain (3Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Crossref PubMed Scopus (732) Google Scholar, 4Busch S.J. Sassone-Corsi P. Trends Genet. 1990; 6: 36-40Abstract Full Text PDF PubMed Scopus (293) Google Scholar). Recently, the human analog of pDIP was characterized by cDNA analysis, showing that human DIP (hDIP) differs from the porcine protein in only four residues. Notably, Arg55 of pDIP is changed to a cysteine in hDIP. hDIP was shown to be present in a large number of tissues by reverse transcription-polymerase chain reaction/Southern hybridization (5Vogel P. Mägert H.W. Cieslak A. Adermann K. Forssmann W.G. Biochim. Biophys. Acta. 1996; 1309: 200-204Crossref PubMed Scopus (18) Google Scholar). pDIP shares significant homology with the murine TSC-22 protein and the product of the Drosophila melanogaster shortsighted (shs)gene, and the regions upstream of the leucine zipper are almost identical. TSC-22 was reported to be present in both the cytoplasmic and the nuclear fraction, and it has been discussed to function as a transcriptional regulator that is activated by transcription growth factor-β1 and other growth factors of osteoblastic cells (6Shibanuma M. Kuroki T. Nose K. J. Biol. Chem. 1992; 267: 10219-10224Abstract Full Text PDF PubMed Google Scholar). TheDrosophila shs gene product acts in the decapentaplegic pathway leading to photoreceptor differentiation (7Treisman J.E. Lai Z.C. Rubin G.M. Development. 1995; 121: 2835-2845PubMed Google Scholar). In contrast to TSC-22, shs gene product is a cytoplasmic protein present anterior to the furrow. The evolutionary conservation suggests the region upstream of the leucine zipper to be a distinct functional or structural domain. The well known leucine zipper motif consists of two α-helices with the same sequential directionality forming a coiled coil. The coiled coil represents one of the most efficient packing modes of helices (8Crick F.H.C. Acta Crystallogr. Sec. D. 1953; 6: 689-697Crossref Google Scholar) and serves as a model for studies of protein stability and subunit interactions (9Talbot J.A. Hodges R.S. Acc. Chem. Res. 1982; 15: 224-230Crossref Scopus (86) Google Scholar, 10Lau S.Y.M. Taneja A.K. Hodges R.S. J. Biol. Chem. 1984; 259: 13253-13261Abstract Full Text PDF PubMed Google Scholar, 11Hodges R.S. Semchuk P.D. Taneja A.K. Kay C.M. Parker J.M.R. Mant C.T. Pept. Res. 1988; 1: 19-30PubMed Google Scholar, 12Zhou N.E. Zhu B.Y. Kay C.M. Hodges R.S. Biopolymers. 1992; 32: 419-426Crossref PubMed Scopus (119) Google Scholar, 13Adamson J.G. Zhou N.E. Hodges R.S. Curr. Biol. 1993; 4: 428-437Google Scholar). The two-stranded α-helical coiled coil is characterized by a heptade repeat denoted as “abcdefg” where positions a and d are usually occupied by large hydrophobic amino acids such as Leu, Ile, and Val (14Cohen C. Parry D.A.D. Trends Biochem. Sci. 1986; 11: 245-248Abstract Full Text PDF Scopus (302) Google Scholar, 15Cohen C. Parry D.A.D. Proteins Struct. Funct. Genet. 1990; 7: 1-15Crossref PubMed Scopus (666) Google Scholar), and positions e and g by oppositely charged amino acids (16McLachlan A.D. Steward M. J. Mol. Biol. 1975; 98: 293-304Crossref PubMed Scopus (568) Google Scholar, 17.Stone, D., Sodek, J., Johnson, P., Smillie, L. B., Proceedings of the IX Federation of European Biochemical Societies Meeting, Proteins of Contractile Systems, Biro, E. N. A., 1975, 125, 136, North Holland, Amsterdam.Google Scholar). Residues at the a and d positions have profound effects on the oligomerization states of coiled coils (18Harbury P.B. Zhang T. Kim P.S. Alber T. Science. 1993; 262: 1401-1407Crossref PubMed Scopus (1334) Google Scholar, 19Harbury P.B. Kim P.S. Alber T. Nature. 1994; 371: 80-83Crossref PubMed Scopus (419) Google Scholar, 20Zhu B.Y. Zhou N.E. Kay C.M. Hodges R.S. Protein Sci. 1993; 2: 383-394Crossref PubMed Scopus (161) Google Scholar). Much attention has been paid recently to the study of coiled coil domains (21Fairman R. Chao H.G. Mueller L. Lavoie T.B. Shen L. Novotny J. Matsueda G.R. Protein Sci. 1995; 4: 1457-1469Crossref PubMed Scopus (88) Google Scholar, 22Junius F.K. Mackay J.P. Bubb W.A. Jensen S.A. Weiss A.S. King G.F. Biochemistry. 1995; 34: 6164-6174Crossref PubMed Scopus (69) Google Scholar, 23Muhle-Goll C. Nilges M. Pastore A. Biochemistry. 1995; 34: 13554-13564Crossref PubMed Scopus (45) Google Scholar, 24Wendt H. Berger C. Baici A. Thomas R.M. Bosshard H.R. Biochemistry. 1995; 34: 4097-4107Crossref PubMed Scopus (156) Google Scholar) or model α-helical peptides (20Zhu B.Y. Zhou N.E. Kay C.M. Hodges R.S. Protein Sci. 1993; 2: 383-394Crossref PubMed Scopus (161) Google Scholar, 25Kohn D.W. Kay C.M. Hodges R.S. Protein Sci. 1995; 4: 237-250Crossref PubMed Scopus (131) Google Scholar, 26Nautiyal S. Woolfson D.N. King D.S. Alber T. Biochemistry. 1995; 34: 11645-11651Crossref PubMed Scopus (129) Google Scholar). bZIP domains were widely studied with a variety of experimental methods (27Saudek V. Pastore A. Morelli M.A.C. Frank R. Gausepohl H. Gibson T. Weih F. Rösch P. Protein Eng. 1990; 4: 3-10Crossref PubMed Scopus (58) Google Scholar,28Ellenberger T.E. Brandl C.J. Struhl K. Harrison S.C. Cell. 1992; 71: 1223-1237Abstract Full Text PDF PubMed Scopus (813) Google Scholar). Here, however, we present experimental and computational data on an acetylated full-length leucine zipper protein not containing a DNA binding basic domain to more completely understand structural features of leucine zipper domains in the context of full-length proteins. Peptides were assembled using Fmoc chemistry on an automated peptide synthesizer (model 9050, PerSeptive Biosystems, Wiesbaden, Germany). Fmoc amino acids were purchased from Orpegen (Heidelberg, Germany) and PerSeptive Biosystems. Fmoc-Arg(Pbf) was from Sygena (Liestal, Switzerland).N,N-Dimethylformamide (peptide synthesis grade) and polyethylene glycol-polystyrine resins were purchased from PerSeptive Biosystems. TBTU was from Peboc (Llangefni, Wales). Acetonitrile (HPLC grade), acetic anhydride, dichloromethane,tert-butylmethyl ether, pyridine, piperidine, 1,2-ethanedithiol, and trifluoroacetic acid were purchased from Merck (Darmstadt, Germany). 1-Hydroxybenzotriazole and diisopropylethylamine were obtained from Fluka (Neu-Ulm, Germany). Solid phase synthesis of pDIP was carried out on a preloaded Fmoc-Val-polyethylene glycol-polystyrine resin (loading 0.19 mmol/g, 0.78 g), while fragment pDIP(9–46) was synthesized using a Fmoc-peptide amide liuber-polyethylene glycol-polystyrene amide resin (loading 0.16 mmol/g, 0.8 g). Acylations were performed in 30 min with a 4-fold excess of Fmoc-l-amino acid in the presence of TBTU/diisopropylethylamine/1-hydroxybenzotriazole (4 eq). Fmoc groups were cleaved by treatment with 20% piperidine inN,N-dimethylformamide for 10 min. After deprotection of the terminal amino groups, the peptidyl resins were acetylated with a mixture of dichloromethane/N,N-dimethylformamide/acetic anhydride/pyridine (40:40:19:1, volume) in 20 min at 0 °C. Subsequently, the resins were washed withN,N-dimethylformamide, 2-propanol, and dichloromethane (3×) and then dried to a constant weight. Resin cleavage and deprotection were carried out with a freshly prepared mixture of trifluoroacetic acid/ethanedithiol/water (94:3:3, volume, 10 ml/g resin) for 2 h. After filtration, the resin was washed with trifluoroacetic acid, and the crude peptide was precipitated by addition of chilled tert-butylmethyl ether, washed with tert-butylmethyl ether, and lyophilized from 5% acetic acid (crude yields: pDIP, 516 mg, 39.7%; pDIP(9–46), 225 mg, 38.7%). For purification, the dried crude products were dissolved in water (50 ml), loaded onto a Vydac C18 column (20 × 250 mm, 10 μm, 300 Å, The Separations Group, Hesperia, CA) and separated (buffer A: 0.06% trifluoroacetic acid in water; buffer B: 0.05% trifluoroacetic acid in acetonitrile/water, 4:1, linear gradient 20–100% buffer B in 80 min, flow rate 9 ml/min, detection at 230 nm). Pure fractions, detected by analytical HPLC (Vydac C18, 5 μm, 300 Å, 4.6 × 250 mm, flow, 0.8 ml/min, detection at 215 nm) were pooled and lyophilized. pDIP: yield, 66 mg (5.1%, calculated from initial resin loading). Molecular weight (M r) by electrospray mass spectroscopy [M + 4H]4+ 2189.5 (M r calculated 8754.9). Amino acid composition (after hydrolysis with 6 n HCl at 150 °C for 90 min, 1090 Aminoquant, Hewlett-Packard): Ala 6.15 (6Shibanuma M. Kuroki T. Nose K. J. Biol. Chem. 1992; 267: 10219-10224Abstract Full Text PDF PubMed Google Scholar), Arg 3.80 (4Busch S.J. Sassone-Corsi P. Trends Genet. 1990; 6: 36-40Abstract Full Text PDF PubMed Scopus (293) Google Scholar), Asx 4.20 (4Busch S.J. Sassone-Corsi P. Trends Genet. 1990; 6: 36-40Abstract Full Text PDF PubMed Scopus (293) Google Scholar), Glx 19.50 (19Harbury P.B. Kim P.S. Alber T. Nature. 1994; 371: 80-83Crossref PubMed Scopus (419) Google Scholar), Gly 2.09 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), His 1.06 (1Monnier M. Dudler L. Gächter R. Maier P.F. Tobler H.J. Schoenenberger G.A. Experimentia (Basel). 1977; 33: 548-552Crossref PubMed Scopus (43) Google Scholar), Ile 1.98 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), Leu 9.92 (10Lau S.Y.M. Taneja A.K. Hodges R.S. J. Biol. Chem. 1984; 259: 13253-13261Abstract Full Text PDF PubMed Google Scholar), Lys 5.09 (5Vogel P. Mägert H.W. Cieslak A. Adermann K. Forssmann W.G. Biochim. Biophys. Acta. 1996; 1309: 200-204Crossref PubMed Scopus (18) Google Scholar), Met 1.99 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), Phe 1.01 (1Monnier M. Dudler L. Gächter R. Maier P.F. Tobler H.J. Schoenenberger G.A. Experimentia (Basel). 1977; 33: 548-552Crossref PubMed Scopus (43) Google Scholar), Pro 7.10 (7Treisman J.E. Lai Z.C. Rubin G.M. Development. 1995; 121: 2835-2845PubMed Google Scholar), Ser 4.69 (5Vogel P. Mägert H.W. Cieslak A. Adermann K. Forssmann W.G. Biochim. Biophys. Acta. 1996; 1309: 200-204Crossref PubMed Scopus (18) Google Scholar), Thr 2.89 (3Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Crossref PubMed Scopus (732) Google Scholar), Tyr 1.05 (1Monnier M. Dudler L. Gächter R. Maier P.F. Tobler H.J. Schoenenberger G.A. Experimentia (Basel). 1977; 33: 548-552Crossref PubMed Scopus (43) Google Scholar), Val 5.01 (5Vogel P. Mägert H.W. Cieslak A. Adermann K. Forssmann W.G. Biochim. Biophys. Acta. 1996; 1309: 200-204Crossref PubMed Scopus (18) Google Scholar). pDIP(9–46): yield, 32.5 mg (5.6%). Electrospray mass spectroscopy, [M + 2H]2+2271.5 (M r calculated 4542.7). Amino acid composition: Ala 2.02 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), Arg 2.82 (3Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Crossref PubMed Scopus (732) Google Scholar), Asx 1.99 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), Glx 10.41 (10Lau S.Y.M. Taneja A.K. Hodges R.S. J. Biol. Chem. 1984; 259: 13253-13261Abstract Full Text PDF PubMed Google Scholar), Ile 1.93 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), Leu 6.11 (6Shibanuma M. Kuroki T. Nose K. J. Biol. Chem. 1992; 267: 10219-10224Abstract Full Text PDF PubMed Google Scholar), Lys 2.99 (3Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Crossref PubMed Scopus (732) Google Scholar), Met 0.96 (1Monnier M. Dudler L. Gächter R. Maier P.F. Tobler H.J. Schoenenberger G.A. Experimentia (Basel). 1977; 33: 548-552Crossref PubMed Scopus (43) Google Scholar), Pro 0.97 (1Monnier M. Dudler L. Gächter R. Maier P.F. Tobler H.J. Schoenenberger G.A. Experimentia (Basel). 1977; 33: 548-552Crossref PubMed Scopus (43) Google Scholar), Ser 1.91 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), Thr 2.07 (2Sillard R. Schulz-Knappe P. Vogel P. Raida M. Bensch W. Forssmann W.G. Mutt V. Eur. J. Biochem. 1993; 216: 429-436Crossref PubMed Scopus (27) Google Scholar), Tyr 1.10 (1Monnier M. Dudler L. Gächter R. Maier P.F. Tobler H.J. Schoenenberger G.A. Experimentia (Basel). 1977; 33: 548-552Crossref PubMed Scopus (43) Google Scholar), Val 2.92 (3Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Crossref PubMed Scopus (732) Google Scholar). Peptides were loaded at 4 μm–2.5 mm on a Superdex 75 column equilibrated in 50 mm potassium phosphate buffer, pH 7, 0.1 m NaSO4, using a flow rate of 0.5 ml/min. The column was calibrated with bovine serum albumin (67 kDa), ovalbumine (43 kDa), chymotrypsinogen A (25 kDa) ribonuclease A (13.7 kDa), ubiquitin (8.5 kDa), and blue dextran 2000 (2 MDa). Sedimentation experiments were performed at room temperature in a Beckman model E analytical ultracentrifuge equipped with a high-intensity light source, 10“ recorder, and AnH-Ti rotor. Measurements under the conditions of the NMR experiments made use of cells with ultra-thin pathlength and schlierenoptics: sedimentation velocity at 68,000 rpm, high-speed sedimentation equilibrium at 30,000 and 24,000 rpm; UV scanning at 240 nm. Initial peptide concentrations were 0.2 and 1.1 mm in 50 mm potassium phosphate buffer, pH 7, and 50 mm potassium phosphate buffer, 100 mmNa2SO4, pH 7, respectively. The partial specific volume was calculated from the amino acid composition. The sedimentation coefficient was calculated from the slope in an ln(r) versus time plot, the molecular mass distribution was fitted assuming a monomer-dimer equilibrium using the „multeq 3b” program kindly provided by Dr. A. Minton. For the determination of the diffusion coefficient, areas and maximal gradients were obtained from 20-fold magnifications of schlieren photographies. CD spectra were measured on a Jasco J600 CD spectropolarimeter. A water bath was used to control the cell temperature. Data were collected with a 0.2 nm step resolution, a time constant of 1 s, and a scan speed of 20 nm/min. Wavelength scans were performed at discrete temperatures from 25 to 90 °C in a thermostatically controlled quartz cell of 0.5 or 0.05 cm pathlength, depending on peptide concentration. For each temperature point, spectra were obtained from 250 to 190 nm. Wavelength scans were processed by subtracting buffer scans taken at the same temperature and converting the data set to mean residual ellipticity prior to analysis. 10 mm phosphate buffer was used throughout. Thermal transition curves were recorded from 5 to 90 or 95 °C at a fixed wavelength of 222 nm. The samples were heated at intervals of 2.5–5 °C. At each temperature the samples were allowed to equilibrate for 15–20 min. The reversibility of the denaturations was verified by slowly cooling the sample from final to initial temperature. Typically, more than 95% of the CD signal was recovered after cooling. The transition curves were normalized to the fraction of the unfolded peptide using the standard equation: f u = (θ222 − θ n )/(θ u − θ n ), where θ n and θ u represent the ellipticity values for the fully folded and fully unfolded species, respectively, and θ222 is the observed ellipticity at 222 nm. The transition temperature, T m , of each heat-induced reversible denaturation was determined by fitting the CD signal change at 222 nm (θ222) as a function of temperature to a two-state denaturation process using a least-squares fit program based on equations described elsewhere (29Bowie J.U. Sauer R.T. Biochemistry. 1989; 28: 7139-7143Crossref PubMed Scopus (221) Google Scholar). The conformational stability ΔG 0 was determined as ΔG 0 = ΔG +RT ln P, where P is the total protein concentration. Secondary structure predictions were carried out using the Gibrat et al. (30Gibrat J.-F. Garnier J. Robson B. J. Mol. Biol. 1987; 198: 425-443Crossref PubMed Scopus (488) Google Scholar), Levin et al. (31Levin J.M. Robson B. Garnier J. FEBS Lett. 1986; 205: 303-308Crossref PubMed Scopus (261) Google Scholar), double prediction method (32Deleage G. Roux B. Protein Eng. 1987; 1: 289-294Crossref PubMed Scopus (319) Google Scholar), self-optimized prediction methods from alignments (33Geourjon C. Deleage G. Protein Eng. 1994; 7: 157-164Crossref PubMed Scopus (308) Google Scholar), profile network from Heidelberg (34Rost B. Sander C. Proteins. 1994; 19: 55-72Crossref PubMed Scopus (1337) Google Scholar), secondary structure prediction (35Mehta P.K. Hering J. Argos P. Protein Sci. 1995; 4: 2517-2525Crossref PubMed Scopus (83) Google Scholar), and PREDATOR (36Frishman D. Argos P. Protein Eng. 1996; 9: 133-142Crossref PubMed Scopus (352) Google Scholar) algorithms. NMR measurements were carried out on a standard Bruker AMX 600 spectrometer with 3.5 mm pDIP in 50 mm potassium phosphate buffer, pH 3.5, 298 K. Data from the following NMR spectra were employed for the sequence specific assignment of spin systems and the evaluation of nuclear Overhauser enhancement spectroscopy (NOESY) distance constraints: double quantum filtered correlated spectroscopy, total correlation spectroscopy with 40, 80, and 120 ms of mixing time, respectively, and a spin lock field of approximately 12 kHz and NOESY with mixing times of 100 and 200 ms, respectively. Solvent suppression was performed by continuous coherent irradiation prior to the first excitation pulse and during the mixing time in the NOESY experiment. The sweep width in ω1 and ω2 was 7246 Hz for all spectra. Quadrature detection was used in both dimensions with the time proportional phase incrementation technique in ω1 (37Marion D. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3520) Google Scholar). 4 K data points were collected in ω2 and 512 data points in ω1. Zero filling of the time domain data resulted in a frequency-domain matrix with 1 and 2 K data points in ω1 and ω2, respectively. All two-dimensional spectra were multiplied by a squared sineball function phase shifted by π/4 for NOESY and for total correlation spectroscopy spectra and by π/8 for COSY spectra. Base-line correction to the seventh order was used. Data were evaluated on X-window workstations with the NDee program package (Software Symbiose, Inc., Bayreuth, Germany). Chemical shift values are reported relative to external 2,2-dimethyl-2-silapentane sulfonate. Distance information was obtained from two-dimensional NOESY spectra in H2O/2H2O (9:1) and in 99.998%2H2O. NOESY cross peaks were classified into three categories according to their volume intensity as estimated from the number of contours in NOESY spectra: strong 0.18–0.27 nm; medium, 0.18–0.4 nm; weak, 1.8–5.5 nm. Pseudoatom corrections were used to adjust distances that involved nonstereospecifically assigned protons such as methyl groups or aromatic ring protons (38Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Three-dimensional structures were calculated with the X-PLOR 3.1 package (39Brünger A.T. XPLOR Version 3.1. Yale University Press, New Haven, CT1993Google Scholar). The standard protocols for ab initio simulated annealing and simulated annealing refinement were applied with some modifications. The initial structure calculations started from an extended template with satisfactory local geometry. For the leucine zipper domain a modeling approach for coiled coil proteins (40DeLano W.L. Brünger A.T. Proteins. 1994; 20: 105-123Crossref PubMed Scopus (53) Google Scholar) was used. The method draws upon knowledge of the oligomerization state, the helix directionality, and the properties of heptade repeat sequences. Unknown structural parameters are heavily sampled. The coiled coil twist angle, for example, is sampled with an initial range from −35° to 35° in one degree increments. The initial Cα positions were those of a regular α-helix, and the initial separation between the helices was set to 10 Å. For each initial structure, side chain and backbone atoms were grown from the Cα position by applying a protocol similar to those used for the generation of initial coordinates in NMR structure determination (41Nilges M. Clore M. Gronenborn A.M. FEBS Lett. 1988; 239: 129-136Crossref PubMed Scopus (524) Google Scholar). Each structure was relaxed with the following simulated annealing protocol: (i) a 5 ps molecular dynamics slow-cooling stage from 500 to 300 K, (ii) a 20 ps constant temperature molecular dynamics (MD) simulation at 300 K, and (iii) 1000 steps of conjugate gradient minimization. During the slow-cooling stage, Cα atoms were held in place with harmonic point restraints that were slowly reduced. The helical hydrogen bond restraints were active during all stages, but no other restraints were applied during the constant temperature MD and energy minimization stages. A time step of 0.5 fs was used for temperatures above 350 K during the slow-cooling stage, otherwise a time step of 1 fs was applied. The coordinates for the residues in the leucine zipper domain thus obtained were used as a reference set in the ab initio simulated annealing and the simulated annealing refinement to restrain the main coordinate set. The restraints were incorporated as point restraints in the form of a harmonic potential. Unrestrained MD calculations were carried out using the parameters for a representative leucine zipper structure and the TIP3P water model (42Jørgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (29453) Google Scholar) that was supplied with the standard X-PLOR force field (39Brünger A.T. XPLOR Version 3.1. Yale University Press, New Haven, CT1993Google Scholar). The overlay was achieved by placing the protein in the center of a cubic water box (6.33 nm) and deleting all solvent molecules closer than 0.26 nm to any heavy atom of the protein. Close nonbonded solute-solvent interactions were removed in two steps. First, 100 cycles of conjugate gradient energy minimization (43Powell M.J.D. Mathemat. Progr. 1977; 12: 241-254Crossref Scopus (1420) Google Scholar) were carried out, keeping the positions of all protein atoms fixed. Second, in 300 cycles of energy minimization, a harmonic potential was used to restrain the protein to its original conformation. During the first 15 ps of the MD calculations, the system was gradually heated to 300 K while coupled to an external water bath (44Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. DiNiola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23131) Google Scholar). The MD calculations were carried out using the Verlet algorithm (45Verlet L. Physiol. Rev. 1967; 159: 98-103Crossref Scopus (6977) Google Scholar) with a time step of 1 fs. The SHAKE algorithm (46van Gunsteren W.F. Berendsen H.J.C. J. Mol. Phys. 1977; 34: 1311-1327Crossref Scopus (1575) Google Scholar) was used to constrain covalent bond lengths. A dielectric constant of 1.0 was applied with a scaling factor of 0.4 for 1–4 electrostatic interactions. All nonbonded interactions were cut off at a distance of 0.85 nm. During the whole simulation of 200 ps, minimum image periodic boundary conditions were used. Coordinates, energies, and velocities were saved every 0.5 ps for further analysis. Simulations and analyses were performed on Hewlett Packard HP 735 computers. A 1-ps simulation required about 3 h of CPU time. A second unrestrained MD calculation was carried out with the leucine zipper of GCN4 using the crystal structure (47O'Shea E.K. Klemm J.
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