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• W1976735107 abstract "The region encompassing residues 181–98 on the α1 subunit of the muscle-type nicotinic acetylcholine receptor forms a major determinant for the binding of α-neurotoxins. We have prepared an 15N-enriched 18-amino acid peptide corresponding to the sequence in this region to facilitate structural elucidation by multidimensional NMR. Our aim was to determine the structural basis for the high affinity, stoichiometric complex formed between this cognate peptide and α-bungarotoxin, a long α-neurotoxin. Resonances in the complex were assigned through heteronuclear and homonuclear NMR experiments, and the resulting interproton distance constraints were used to generate ensemble structures of the complex. Thr8, Pro10, Lys38, Val39, Val40, and Pro69 in α-bungarotoxin and Tyr189, Tyr190, Thr191, Cys192, Asp195, and Thr196 in the peptide participate in major intermolecular contacts. A comparison of the free and bound α-bungarotoxin structures reveals significant conformational rearrangements in flexible regions of α-bungarotoxin, mainly loops I, II, and the C-terminal tail. Furthermore, several of the calculated structures suggest that cation-π interactions may be involved in binding. The root mean square deviation of the polypeptide backbone in the complex is 2.07 Å. This structure provides, to date, the highest resolution description of the contacts between a prototypic α-neurotoxin and its cognate recognition sequence. The region encompassing residues 181–98 on the α1 subunit of the muscle-type nicotinic acetylcholine receptor forms a major determinant for the binding of α-neurotoxins. We have prepared an 15N-enriched 18-amino acid peptide corresponding to the sequence in this region to facilitate structural elucidation by multidimensional NMR. Our aim was to determine the structural basis for the high affinity, stoichiometric complex formed between this cognate peptide and α-bungarotoxin, a long α-neurotoxin. Resonances in the complex were assigned through heteronuclear and homonuclear NMR experiments, and the resulting interproton distance constraints were used to generate ensemble structures of the complex. Thr8, Pro10, Lys38, Val39, Val40, and Pro69 in α-bungarotoxin and Tyr189, Tyr190, Thr191, Cys192, Asp195, and Thr196 in the peptide participate in major intermolecular contacts. A comparison of the free and bound α-bungarotoxin structures reveals significant conformational rearrangements in flexible regions of α-bungarotoxin, mainly loops I, II, and the C-terminal tail. Furthermore, several of the calculated structures suggest that cation-π interactions may be involved in binding. The root mean square deviation of the polypeptide backbone in the complex is 2.07 Å. This structure provides, to date, the highest resolution description of the contacts between a prototypic α-neurotoxin and its cognate recognition sequence. nicotinic acetylcholine receptor α-bungarotoxin high performance liquid chromatography cyanogen bromide heteronuclear single quantum correlation total correlation spectroscopy nuclear Overhauser effect nuclear Overhauser enhancement spectroscopy root mean square deviation Naja mossambica mossambica I The nicotinic acetylcholine receptor (nAChR)1 (1Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar) has long been a prototype for ligand-gated ion channels. This receptor is involved in excitatory synaptic transmission at the neuromuscular junction and also plays an important role in the nervous system. The nAChRs are pentameric complexes composed of homologous subunits with subunits arranged around the central channel in a symmetrical manner. The muscle-type nAChR contains two α1 subunits and one each of the β1, γ(ε), and δ subunits. The ligand binding sites are situated at the αγ(ε) and αδ subunit interfaces. The muscle-type nAChR serves as an important model for the study of the structures and functions of related ligand-gated ion channels (for review, see Ref.1Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar). The snake venom-derived α-neurotoxins fall into two categories, short and long neurotoxins, and act as high affinity competitive antagonists at the nAChR. Short neurotoxins (e.g. erabutoxina) contain 60–62 amino acid residues and 4 conserved disulfide bridges. Long neurotoxins have 66–74 residues and 5 disulfide bonds, including four in a core region that are homologous in position to those found in the short neurotoxins. α-Bungarotoxin (Bgtx), obtained from the snake venom of Bungarus multicinctus, is a long α-neurotoxin that over the years has provided a powerful tool for the study of muscle-type nAChRs and which has come to be viewed as somewhat of a gold standard among the α-neurotoxins. A number of the α-neurotoxins have been heterologously expressed in recent years, allowing for investigations using site-directed mutagenesis (2Antil S. Servent D. Ménez A. J. Biol. Chem. 1999; 274: 34851-34858Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Malany S. Osaka H. Sine S.M. Taylor P. Biochemistry. 2000; 39: 15388-15398Crossref PubMed Scopus (32) Google Scholar, 4Rosenthal J.A. Levandoski M.M. Chang B. Potts J.F. Shi Q.-L. Hawrot E. Biochemistry. 1999; 38: 7847-7855Crossref PubMed Scopus (34) Google Scholar, 5Rosenthal J.A. Hsu S.H. Schneider D. Gentile L.N. Messier N.J. Vaslet C.A. Hawrot E. J. Biol. Chem. 1994; 269: 11178-11185Abstract Full Text PDF PubMed Google Scholar). From its x-ray structure, Bgtx is a relatively flat, slightly concave, disc-shaped protein with a characteristic three-finger folding motif consisting of three loops of structure (6Love R.A. Stroud R.M. Protein Eng. 1986; 1: 37-46Crossref PubMed Scopus (155) Google Scholar). Previous NMR structural studies indicate that the solution structure of Bgtx, although generally consistent with the x-ray structure, does reveal some differences (7Basus V.J. Billeter M. Love R.A. Stroud R.M. Kuntz I.D. Biochemistry. 1988; 27: 2763-2771Crossref PubMed Scopus (78) Google Scholar). Notably, the side chain of Trp28 in the two structures resides on opposite sides of the major plane of the molecule. In the solution structure, the Trp side chain is on the concave surface, as seen with most other α-neurotoxins containing this highly conserved residue. In contrast, the Trp side chain is located on the opposite face in the crystal structure (6Love R.A. Stroud R.M. Protein Eng. 1986; 1: 37-46Crossref PubMed Scopus (155) Google Scholar). The structures of several other snake venom α-neurotoxins have been studied with NMR techniques (8Labhardt A.M. Hunziker-Kwik E.H. Wüthrich K. Eur. J. Biochem. 1988; 177: 295-305Crossref PubMed Scopus (38) Google Scholar, 9Le-Goas R. LaPlante S.R. Mikou A. Delsuc M.A. Guittet E. Robin M. Charpentier I. Lallemand J.Y. Biochemistry. 1992; 31: 4867-4875Crossref PubMed Scopus (51) Google Scholar, 10Zinn-Justin S. Roumestand C. Gilquin B. Bontems F. Ménez A. Toma F. Biochemistry. 1992; 31: 11335-11347Crossref PubMed Scopus (87) Google Scholar, 11Connolly P.J. Stern A.S. Hoch J.C. Biochemistry. 1996; 35: 418-426Crossref PubMed Scopus (19) Google Scholar, 12Peng S.-S. Kumar T.K.S. Jayaraman G. Chang C.-C. Yu C. J. Biol. Chem. 1997; 272: 7817-7823Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), and all exhibit the characteristic three-finger structure. Previous work indicates that the main determinants for Bgtx binding to the muscle-type nAChR lie between residues 173 and 204 of the α1 subunit (13Wilson P.T. Lentz T.L. Hawrot E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8790-8794Crossref PubMed Scopus (121) Google Scholar), a region that coincides with one of three segments of the α subunit that have been implicated in agonist binding (for review, see Ref. 1Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar). Tyr190, along with Cys192, Cys193, and Tyr198 are selectively cross-linked with a variety of site-directed photoaffinity reagents (14Grutter T. Ehret-Sabatier L. Kotzyba-Hibert F. Goeldner M. Biochemistry. 2000; 39: 3034-3043Crossref PubMed Scopus (21) Google Scholar). This region, termed segment C, contains a conserved pair of adjacent Cys residues, Cys192-Cys193, that form an unusual disulfide. Studies of synthetic peptides with sequences matching those in segment C have identified several peptides that bind Bgtx with affinities in the micromolar to submicromolar range (15Kachalsky S.G. Jensen B.S. Barchan D. Fuchs S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10801-10805Crossref PubMed Scopus (37) Google Scholar, 16Wilson P.T. Hawrot E. Lentz T.L. Mol. Pharmacol. 1988; 34: 643-650PubMed Google Scholar). A peptide fragment (α18-mer) with a sequence corresponding to amino acid residues 181–198 (α-Y181RGWKHWVYYTCCPDTPY198) from theTorpedo californica nAChR binds Bgtx with an apparentKD of ∼65 nm (17Pearce S.F.A. Preston-Hurlburt P. Hawrot E. Proc. R. Soc. Lond. Biol. Sci. 1990; 241: 207-213Crossref PubMed Scopus (20) Google Scholar). Replacing the Tyr at position 190 with a Phe leads to a 60-fold decrease in Bgtx binding affinity for the altered peptide, suggesting an important role for this aromatic residue in complex formation (17Pearce S.F.A. Preston-Hurlburt P. Hawrot E. Proc. R. Soc. Lond. Biol. Sci. 1990; 241: 207-213Crossref PubMed Scopus (20) Google Scholar). Mutations of Tyr190, when assessed in heterologous expression systems, also result in large decreases in α-neurotoxin binding (3Malany S. Osaka H. Sine S.M. Taylor P. Biochemistry. 2000; 39: 15388-15398Crossref PubMed Scopus (32) Google Scholar, 18Spura A. Riel R.U. Freedman N.D. Agrawal S. Seto C. Hawrot E. J. Biol. Chem. 2000; 275: 22452-22460Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 19Ackermann E.J. Ang E.T.-H. Kanter J.R. Tsigelny I. Taylor P. J. Biol. Chem. 1998; 273: 10958-10964Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In addition, ligand gating is also dramatically affected by mutations in Tyr190 (20Tomaselli G.F. McLaughlin J.T. Jurman M.E. Hawrot E. Yellen G. Biophys. J. 1991; 60: 721-727Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 21Galzi J.L. Bertrand D. Devillers-Thiery A. Revah F. Bertrand S. Changeux J.P. FEBS Lett. 1991; 294: 198-202Crossref PubMed Scopus (137) Google Scholar). Studies with recombinant receptor fragments corresponding to the α subunit from the mongoose nAChR, which is resistant to α-neurotoxins, suggest two subsites in the binding domain for Bgtx; one is a proline subsite consisting of Pro194 and Pro197, and the other is an aromatic subsite involving positions 187 and 189 (15Kachalsky S.G. Jensen B.S. Barchan D. Fuchs S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10801-10805Crossref PubMed Scopus (37) Google Scholar). We previously described some of the structural features revealed by an NMR analysis of Bgtx complexed with a 12-amino acid peptide fragment (α12-mer) of the Torpedo nAChR α1 subunit (α-K185HWVYYTCCPDT196), which has an apparentKD of ∼1.4 μm (17Pearce S.F.A. Preston-Hurlburt P. Hawrot E. Proc. R. Soc. Lond. Biol. Sci. 1990; 241: 207-213Crossref PubMed Scopus (20) Google Scholar, 22Basus V.J. Song G. Hawrot E. Biochemistry. 1993; 32: 12290-12298Crossref PubMed Scopus (95) Google Scholar). We now describe a more expansive NMR structural analysis of the higher affinity complex formed with the α18-mer. The structure of this complex may provide valuable information on the orientation of the contact residues in the native nAChR and may help in elucidating the essential interactions that direct the ability of the α-neurotoxins to recognize receptor sequences with remarkable affinity and specificity. We designed an oligonucleotide sequence to encode for residues 181–198 (YRGWKHWVYYTCCPDTPY) of the α1 subunit from the nAChR of T. californica. Three copies of this expression cassette were inserted downstream of a 125-amino acid ketosteroid isomerase gene and upstream of a His-tag sequence in plasmid pET-31b(+) (23Kuliopulos A. Walsh C.T. J. Am. Chem. Soc. 1994; 116: 4599-4607Crossref Scopus (124) Google Scholar). The final construct, ketosteroid isomerase-Met-(α18-mer-Met)3-His-tag, contained single Met residues separating the three cassettes from each other, from ketosteroid isomerase, and from the C-terminal His tag. The oligonucleotide sequence of this construct is available upon request. The plasmid, whose insert sequence was confirmed by DNA sequence analysis, was used to transform cells of the expression strain BL21 (DE3) (Novagen). Cell cultures were grown in standard M9 medium except that 15NH4Cl was used as a replacement for normal NH4Cl. All cultures were supplemented with 100 μg/ml ampicillin (M9/Amp). A single colony from a fresh agar plate containing ampicillin was used to inoculate 100 ml M9/Amp medium and grown overnight at 37 °C. The overnight culture was added to 2 liters of M9/Amp medium in a VirTis benchtop fermentor. This culture was grown at 37 °C with stirring at 600 rpm until theA600 was 0.7–0.8. Isopropyl-β-d-thiogalactoside was added to a concentration of 1 mm to initiate induction of the fusion protein. After 3 h, cells were harvested by low speed centrifugation. After resuspension in 40 ml of 20 mm Tris-HCl buffer (pH 7.9), cells were passed through a French pressure cell (SLM Instruments) at 15,000 p.s.i. Inclusion bodies were isolated by centrifugation, resuspended in “binding buffer” (6 m guanidine-HCl, 0.5 mNaCl, 5 mm imidazole, and 20 mm Tris-HCl (pH 7.9)), and applied to a column containing Ni2+-charged His-Bind resin (Novagen) prepared according to the manufacturer's specifications. After washing the resin with 10 column volumes of binding buffer and 5 column volumes of wash buffer (6 mguanidine-HCl, 40 mm imidazole, 0.5 m NaCl, and 20 mm Tris-HCl pH 7.9), the ketosteroid isomerase fusion protein was eluted in 5 column volumes of elution buffer (6m guanidine-HCl, 0.3 m imidazole, 0.5m NaCl, and 20 mm Tris-HCl (pH 7.9)). The fusion protein prepared as described above was dialyzed against water, and the insoluble fusion protein was isolated by centrifugation. The pellet was then resuspended in 20 ml of 80% formic acid in a round-bottom flask and mixed with 1 g of cyanogen bromide (CNBr). After flushing the solution with helium, the flask was sealed, and the reaction was allowed to proceed in the dark for 24 h. The reaction mixture was then diluted 1:1 with water and applied to a C18 Sep-Pak cartridge (Waters). The peptide was eluted with 4 ml of 35% acetonitrile in water and dried using a SpeedVac (Savant). The dry peptide was resuspended in 50 mmsodium phosphate buffer (pH 6.0) at 37 °C for 2 days to deformylate the products. The peptide sample prepared as described above was diluted 1:1 with 0.1% trifluoroacetic acid (buffer A) and applied to a C18 reverse phase Discovery column (Supelco). The peptides were eluted isocratically at 1 ml/min with buffer B (25% acetonitrile with 0.1% trifluoroacetic acid). Peak fractions were collected and then dried using a SpeedVac (Savant). Isolated peptides were analyzed by mass spectrometry (Yale Cancer Center Mass Spectrometry Resource and W. M. Keck Foundation Biotechnology Resource Laboratory). The disulfide form of the peptide with a C-terminal homoserine lactone was chosen for further structural analysis. The15N-labeled disulfide homoserine lactone form of the Torpedo α18-mer was resuspended in 50 mm perdeuterated potassium acetate buffer (pH 4.0) with 5% D2O and 0.05% sodium azide. Bgtx (from Sigma) was prepared in the same buffer at a concentration of 5 mm. Bgtx from this stock solution was added to the α18-mer to form a 1:1 Bgtx·α18-mer complex. The final concentration of the Bgtx·α18-mer complex was 2.1 mm. The “free Bgtx” NMR sample was diluted from stock Bgtx into the same buffer to a final concentration of 2.0 mm. All NMR spectra were recorded on a Bruker Avance 600-MHz NMR spectrometer at a temperature of 35 °C. Chemical shifts at this temperature were calibrated with respect to internal 3-(trimethylsilyl) tetradeutero sodium propionate (0.0 ppm). The formation of the Bgtx·α18-mer complex was followed in a mole-ratio titration using a two-dimensional 15N heteronuclear single quantum correlation (1H-15N HSQC) (24Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-666Crossref PubMed Scopus (3539) Google Scholar, 25Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. Ser. A. 1993; 102: 241-245Crossref Scopus (1116) Google Scholar, 26Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-189Crossref Scopus (2435) Google Scholar) experiment. Amino acid spin systems were identified by two-dimensional total correlation spectroscopy (TOCSY) (24Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-666Crossref PubMed Scopus (3539) Google Scholar, 25Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. Ser. A. 1993; 102: 241-245Crossref Scopus (1116) Google Scholar, 27Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) and three-dimensional TOCSY-HSQC experiments (27Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar, 28Davis A.L. Keeler J. Laue E.D. Moskau D. J. Magn. Reson. 1992; 98: 207-216Google Scholar, 29Palmer III A.G. Cavanagh J. Wright P.E. Rance M. J. Magn. Reson. 1991; 93: 151-170Google Scholar, 30Kay L.E. Keifer P. Saarinen T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2439) Google Scholar, 31Schleucher J. Sattler M. Griesinger C. Angew. Chem. Int. Ed. Engl. 1993; 32: 1489-1491Crossref Scopus (224) Google Scholar) with a mixing time of 60 ms. The assignments of the NH protons and CαH protons of the amino acid spin systems of the peptide were further confirmed by a three-dimensional HNHA experiment (32Vuister G.W. Bax A. J. Am. Chem. Soc. 1993; 115: 7772-7777Crossref Scopus (1055) Google Scholar, 33Vuister G.W. Bax A. J. Biomol. NMR. 1994; 4: 193-200Crossref PubMed Scopus (54) Google Scholar). The three-dimensional HNHA experiment provides the correlation between the 15NH proton and the CαH proton of the same amino acid; these data help confirm the identification of the NH and CαH protons. Nuclear Overhauser effect (NOE) correlations (sequential, medium-range, and long range NOEs) were identified by two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) (24Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-666Crossref PubMed Scopus (3539) Google Scholar, 25Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. Ser. A. 1993; 102: 241-245Crossref Scopus (1116) Google Scholar) and three-dimensional NOESY-HSQC experiments (28Davis A.L. Keeler J. Laue E.D. Moskau D. J. Magn. Reson. 1992; 98: 207-216Google Scholar, 29Palmer III A.G. Cavanagh J. Wright P.E. Rance M. J. Magn. Reson. 1991; 93: 151-170Google Scholar, 30Kay L.E. Keifer P. Saarinen T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2439) Google Scholar, 31Schleucher J. Sattler M. Griesinger C. Angew. Chem. Int. Ed. Engl. 1993; 32: 1489-1491Crossref Scopus (224) Google Scholar) with a mixing time of 120 ms. Spectra from these experiments were also collected at 25 °C to facilitate the assignment of resonances. All NMR spectra were processed and analyzed with XwinNmr (Bruker), NMRPipe (34Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar), and SPARKY (35Goddard T.D. Kneller D.G. SPARKY 3.95. University of California, San Francisco, CA2000Google Scholar). In comparing our results with earlier, more preliminary assignments involving an unlabeled α18-mer peptide bound to Bgtx (36Gentile L.N. Basus V.J. Shi Q.-L. Hawrot E. Ann. N. Y. Acad. Sci. 1995; 757: 222-237Crossref PubMed Scopus (9) Google Scholar), we found that most Bgtx assignments are the same or similar (chemical shifts change less than 0.05 ppm) after calibration. However, the assignments of Val2, His4, Ser9, Ile11, Lys26, Cys29, Cys33, Val40, Lys51, Lys52, Lys70, Gln71, Arg72, and Gly74 were significantly different; in most cases no comparable resonances were observed in our two-dimensional NOESY spectra. On the other hand, new resonance peaks appear elsewhere in the spectra. All these new resonances involving these residues were re-assigned based on sequential NOE connectivity. We think it most likely that the observed chemical shift differences between the two samples are caused by a difference in the ionic strength of the two samples even though both were prepared at pH 4.0 and spectra were acquired at 35 °C. Our present sample is dissolved in 50 mm potassium acetate, whereas the earlier sample was simply adjusted to pH 4.0 with the addition of HCl. A change in the ionic environment could have significant effects on electrostatic interactions between side chains of charged residues, leading to changes in the chemical environment of a subset of spins. Similarly, it was necessary to re-assign most of the α18-mer peptide resonances. Of the eight previously assigned peptide residues, only Asp195and Thr196 are unchanged between the two samples. However, the CαH proton and CβH proton of Thr196 were erroneously assigned previously (36Gentile L.N. Basus V.J. Shi Q.-L. Hawrot E. Ann. N. Y. Acad. Sci. 1995; 757: 222-237Crossref PubMed Scopus (9) Google Scholar). The new swapped assignments incorporate the results from the three-dimensional HNHA experiment. The cross-peak volumes in the two-dimensional NOESY spectra were integrated by the Gaussian fitting protocol using SPARKY. The cross-peaks were classified into three categories: strong, medium, and weak, with corresponding distance ranges of 1.8–3.0, 1.8–4.0, and 1.8–5.0 Å, respectively. The HN-Hα3 J coupling constants of the α18-mer peptide were obtained from the three-dimensional HNHA experiment (37Bax A. Vuister G.W. Grzesiek S. Delaglio F. Wang A.C. Tschudin R. Zhu G. Methods Enzymol. 1994; 239: 79-105Crossref PubMed Scopus (381) Google Scholar). The 3 Jcoupling constants were converted to dihedral angle restraints using previously described methods (38Thompson G.S. Leung Y.-C. Ferguson S.J. Radford S.E. Redfield C. Protein Sci. 2000; 9: 846-858Crossref PubMed Scopus (19) Google Scholar). For 3 J< 6 Hz, the dihedral angle restraint was assigned to −60° ± 30°; for 3 J > 8 Hz, the dihedral angle restraint was −120° ± 40°. All structures were calculated with distance geometry and simulated annealing protocols using the dg_sa.inp script of the NMR structure calculation program, CNSsolve (39Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). The following is the potential energy function used in these calculations:Ftotal = Fbon +Fang + Fimp +Fvdw + Fnoe +Fcdih, where Fbon relates to bond length, Fang andFimp to bond angles, Fvdwrelates to the van der Waals repulsion term,Fnoe relates to NOE distance constraints, andFcdih relates to dihedral angles. Pseudoatoms were used for protons that could not be stereospecifically assigned. The pseudoatom correction feature of CNSsolve was used to adjust the NOE distance constraint range automatically. In each batch of calculations, a different random seed number was used to initiate the calculation of a set of 50 structures. From the pool of calculated structures, only those structures lacking any NOE violation (>0.5 Å) were selected as “acceptable” for further analysis. As a result of the weighting of the Fnoe term in CNSsolve, none of the other energy terms were as critical asFnoe in determining an acceptable structure. In total, 120 acceptable structures of the Bgtx·α18-mer complex were obtained from 6 batches of independent calculations (i.e.300 total structures), and 122 acceptable structures of free Bgtx were obtained from 8 batches of independent calculations (i.e.400 total structures). The 20 lowest-energy structures out of the acceptable structures for free Bgtx and for the Bgtx·α18-mer complex were selected to form an ensemble of representative final structures. The mean structure corresponding to each ensemble was calculated by a program written by Dr. Christian Rölz (40Rölz C. Mierke D.F. Biophys. Chem. 2001; 89: 119-128Crossref PubMed Scopus (14) Google Scholar). The two mean structures (free Bgtx and Bgtx·α18-mer complex) were further partially energy-minimized using DISCOVER (Molecular Simulations, Inc.) to create representative structures complete with side chains. All structures depicted in Fig. 7 have been deposited into the Protein Data Bank, Research Collaboratory for Structural Bioinformatics. The four files corresponding to the mean and ensemble structures for the Bgtx·α18-mer complex and for free Bgtx have been assigned the identifiers 1IDG, 1IDH, 1IDI, 1IDL. We used Rasmol (41Sayle R.A. Milner-White E.J. Trends Biochem. Sci. 1995; 20: 374-376Abstract Full Text PDF PubMed Scopus (2323) Google Scholar), MOLMOL (42Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar), and INSIGHT II (Molecular Simulations, Inc.) for the graphical analysis of the calculated structures. The surface charge potentials were calculated using MOLMOL. The contact surface areas of all the final individual Bgtx·α18-mer complex structures were calculated by contacts of structural units (CSU) using CSU software (43Sobolev V. Sorokine A. Prilusky J. Abola E.E. Edelman M. Bioinformatics. 1999; 15: 327-332Crossref PubMed Scopus (708) Google Scholar). The energetically significant cation-π interaction analysis of the Bgtx·α18-mer complex structures was performed using the CaPTURE program (44Gallivan J.P. Dougherty D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9459-9464Crossref PubMed Scopus (1714) Google Scholar). To facilitate the assignment of the α18-mer peptide resonances while complexed with Bgtx, the peptide corresponding to Torpedo nAChR α1 subunit residues 181–198 was expressed heterologously in Escherichia coli as part of an insoluble fusion protein under conditions where15NH4Cl was used as sole source of nitrogen. After isolation of the fusion protein, CNBr cleavage at engineered Met sites was used to release the desired peptide, which contained the 18 residues of α1 subunit with an additional C-terminal residue derived from the engineered Met. As expected for CNBr cleavage of Met sites, the peptides isolated are a mix of the C-terminal homoserine form of the peptide and its corresponding dehydrated homoserine lactone form. HPLC analysis revealed three major peptide peaks which were further characterized (Fig. 1). Preliminary solid-phase binding studies indicated that all three peptide fractions bind Bgtx to an extent comparable with that obtained with a similar synthetic α18-mer peptide lacking the C-terminal homoserine (data not shown). All three peptide fractions were resistant to thiol alkylation with N-ethylmaleimide except after prior incubation of the peptide with dithiothreitol. These results suggest that the adjacent cysteines, Cys192 and Cys193, are in the disulfide state in the isolated peptides. Mass spectrometric analysis revealed that P2 corresponds to the C-terminal homoserine lactone form of the α18-mer, whereas P1 is the C-terminal homoserine form. The P2 peptide was chosen for the production of a Bgtx·α18-mer complex. The pure α18-mer (P2) was resuspended in 50 mm perdeuterated potassium acetate buffer (pH 4.0) and analyzed with a two-dimensional 1H-15N HSQC experiment that is designed to acquire signal only from protons bound to 15N (Fig. 2 A). An equimolar amount of Bgtx was then added, and the sample was again analyzed by HSQC (Fig. 2 B). A comparison of these spectra (Fig. 2) clearly demonstrates the formation of a stoichiometric complex between the α18-mer and Bgtx. The free peptide (Fig. 2 A) appears to be largely unstructured; all the NH resonances are poorly dispersed in chemical shift and vary in intensity. In contrast, after binding to Bgtx (Fig. 2 B), nearly all the NH resonances undergo large chemical shift changes, and there is little evidence of any free peptide remaining based on the disappearance of resonances seen in the free peptide. These observations suggest that the α18-mer adopts a defined structure upon binding to Bgtx. Furthermore, in mole-ratio titration studies with less than stoichiometric concentrations of Bgtx, NH resonances corresponding to both the bound and the free peptide are present, and the chemical shift of the NH resonances for the bound peptide are fixed and do not vary with Bgtx concentrations (data not shown). These results indicate that the Bgtx·α18-mer complex is in slow exchange. Because only the peptide is15N-enriched, 15N three-dimensional NMR experiments can be used to filter out Bgtx proton signals that are not correlated to 15N. Making use of this enrichment, three-dimensional TOCSY-HSQC, NOESY-HSQC, and HNHA experiments were obtained to make preliminary amino acid assignments of the α18-mer in its bound form. Fig. 3 illustrates a representative strip analysis used to identify the resonances of Lys185. Three-dimensional TOCSY-HSQC analysis is used to identify the resonances correlated by through-bound scalar connectivity to the 15NH (i.e. CαH proton, CβH proton, etc.). Using these three-dimensional NMR experiments, we assigned the observable resonances for all of the amino acid residues in the α18-mer except for the N-terminal Tyr181, which has an exchangeable NH, the C-terminal homoserine lactone, whose mobility may cause its signals to be too weak to be identified, and the two prolines, which lack amide protons (Fig.2 B). These assignments of the peptide resonances greatly facilitated the assignment of the Bgtx resonances in the homonuclear two-" @default.
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• W1976735107 creator A5061582756 @default.
• W1976735107 date "2001-06-01" @default.
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• W1976735107 title "The Solution Structure of the Complex Formed between α-Bungarotoxin and an 18-mer Cognate Peptide Derived from the α1 Subunit of the Nicotinic Acetylcholine Receptor from Torpedo californica" @default.
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