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NMR Structural Analysis of α-Bungarotoxin and Its Complex with the Principal α-Neurotoxin-binding Sequence on the α7 Subunit of a Neuronal Nicotinic Acetylcholine Receptor

2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês

10.1074/jbc.m110320200

ISSN

1083-351X

Autores

Leonard Moise, Andrea Piserchio, Vladimir J. Basus, Edward Hawrot,

Tópico(s)

Ion channel regulation and function

Resumo

We report a new, higher resolution NMR structure of α-bungarotoxin that defines the structure-determining disulfide core and β-sheet regions. We further report the NMR structure of the stoichiometric complex formed between α-bungarotoxin and a recombinantly expressed 19-mer peptide (178IPGKRTESFYECCKEPYPD196) derived from the α7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in α-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the α7 19-mer hairpin-like structure. At the contact zone, Ala7, Ser9, and Ile11 in finger I and Arg36, Lys38, Val39, and Val40 in finger II of α-bungarotoxin interface with Phe186, Tyr187, Glu188, and Tyr194 in the α7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the α7 receptor, places α-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of α-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity. We report a new, higher resolution NMR structure of α-bungarotoxin that defines the structure-determining disulfide core and β-sheet regions. We further report the NMR structure of the stoichiometric complex formed between α-bungarotoxin and a recombinantly expressed 19-mer peptide (178IPGKRTESFYECCKEPYPD196) derived from the α7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in α-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the α7 19-mer hairpin-like structure. At the contact zone, Ala7, Ser9, and Ile11 in finger I and Arg36, Lys38, Val39, and Val40 in finger II of α-bungarotoxin interface with Phe186, Tyr187, Glu188, and Tyr194 in the α7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the α7 receptor, places α-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of α-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity. The nicotinic acetylcholine receptor (nAChR) 1The abbreviations used are: nAChRnicotinic acetylcholine receptorBgtxα-bungarotoxinCbtxα-cobratoxinLSIIILaticauda semifasciata IIINmmIN. mossambica mossambica IAChBPacetylcholine-binding proteinRP-HPLCreverse phase high performance liquid chromatographyDEPTdistortionless enhancement by polarization transferDQF-COSYdouble-quantum-filtered correlation spectroscopyE-COSYexclusive correlation spectroscopyTOCSYtotal correlation spectroscopyNOESYnuclear Overhauser enhancement spectroscopyNOEnuclear Overhauser effectHSQCheteronuclear single quantum correlation3 JHNαscalar coupling constant between HN and HαH/Dhydrogen/deuteriumr.m.s.d.root mean square deviationDSSPdefinitions of the secondary structures of proteins 1The abbreviations used are: nAChRnicotinic acetylcholine receptorBgtxα-bungarotoxinCbtxα-cobratoxinLSIIILaticauda semifasciata IIINmmIN. mossambica mossambica IAChBPacetylcholine-binding proteinRP-HPLCreverse phase high performance liquid chromatographyDEPTdistortionless enhancement by polarization transferDQF-COSYdouble-quantum-filtered correlation spectroscopyE-COSYexclusive correlation spectroscopyTOCSYtotal correlation spectroscopyNOESYnuclear Overhauser enhancement spectroscopyNOEnuclear Overhauser effectHSQCheteronuclear single quantum correlation3 JHNαscalar coupling constant between HN and HαH/Dhydrogen/deuteriumr.m.s.d.root mean square deviationDSSPdefinitions of the secondary structures of proteins (1.Karlin A. Akabas M.H. Neuron. 1995; 15: 1331-1344Abstract Full Text PDF Scopus (563) Google Scholar) is a ligand-gated ion channel that mediates excitatory transmission at the neuromuscular junction and at synapses in the central and peripheral nervous systems. It is the most intensely studied member of the ligand-gated ion channel superfamily and serves as a model for understanding the structure and function of related ion conducting channels including glycine, γ-aminobutyric acid type A, γ-aminobutyric acid type C, and type 3 serotonin receptors. nAChRs are pentameric complexes that assemble in the membrane with 5-fold symmetry. Each subunit contains an N-terminal extracellular domain about 200 amino acids long followed by four membrane-spanning segments (M1–M4) with an intracellular loop of variable length between M3 and M4. The second transmembrane region from each subunit contributes to the formation of the wall lining the channel pore. In muscle and Torpedo electric organ, the subunit composition is (α1)2βγδ and (α1)2βεδ in embryonic and adult tissue, respectively (for review, see Ref. 1.Karlin A. Akabas M.H. Neuron. 1995; 15: 1331-1344Abstract Full Text PDF Scopus (563) Google Scholar). Neuronal nAChR subunits (α2-α10 and β2-β4) apparently can assemble in various combinations giving rise to multiple receptor subtypes. In heterologous expression systems, particular subunit combinations form functional hetero-pentamers (e.g.(α4)2(β2)3) whereas the α7-α9 subunits form functional homo-pentamers (2.Sargent P. Annu. Rev. Neurosci. 1993; 16: 403-443Crossref PubMed Scopus (934) Google Scholar, 3.Grutter T. Changeux J.-P. Trends Biochem. Sci. 2001; 26: 459-462Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). nicotinic acetylcholine receptor α-bungarotoxin α-cobratoxin Laticauda semifasciata III N. mossambica mossambica I acetylcholine-binding protein reverse phase high performance liquid chromatography distortionless enhancement by polarization transfer double-quantum-filtered correlation spectroscopy exclusive correlation spectroscopy total correlation spectroscopy nuclear Overhauser enhancement spectroscopy nuclear Overhauser effect heteronuclear single quantum correlation scalar coupling constant between HN and Hα hydrogen/deuterium root mean square deviation definitions of the secondary structures of proteins nicotinic acetylcholine receptor α-bungarotoxin α-cobratoxin Laticauda semifasciata III N. mossambica mossambica I acetylcholine-binding protein reverse phase high performance liquid chromatography distortionless enhancement by polarization transfer double-quantum-filtered correlation spectroscopy exclusive correlation spectroscopy total correlation spectroscopy nuclear Overhauser enhancement spectroscopy nuclear Overhauser effect heteronuclear single quantum correlation scalar coupling constant between HN and Hα hydrogen/deuterium root mean square deviation definitions of the secondary structures of proteins Snake venom-derived α-neurotoxins bind the muscle-type and, in some cases, homo-pentameric neuronal nAChRs with high affinity (4.Chiapinelli V.A. Harvey A. Natural and Synthetic Neurotoxins. Academic Press, New York1993: 65-128Google Scholar, 5.Servent D. Winckler D.V. Hu H.Y. Kessler P. Drevet P. Bertrand D. Ménez A. J. Biol. Chem. 1997; 272: 24279-24286Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and are grouped into two families (4.Chiapinelli V.A. Harvey A. Natural and Synthetic Neurotoxins. Academic Press, New York1993: 65-128Google Scholar). Short chain neurotoxins have 60–62 amino acids. Long chain toxins have 66–74 amino acids and a fifth disulfide at the tip of the second loop. Solution NMR and x-ray crystallographic studies (e.g. Refs. 6.Zinn-Justin S. Roumestand C. Gilquin B. Bontems F. Ménez A. Toma F. Biochemistry. 1992; 31: 11335-11347Crossref PubMed Scopus (87) Google Scholar, 7.Leroy E. Mikou A. Yang Y. Guittet E. J. Biomol. Struct. Dyn. 1994; 12: 1-17Crossref PubMed Scopus (5) Google Scholar, 8.Betzel C. Lange G. Pal G.P. Wilson K.S. Maelicke A. Saenger W. J. Biol. Chem. 1991; 266: 21530-21536Abstract Full Text PDF PubMed Google Scholar) show that all α-neurotoxins share a tertiary structure known as the three-finger fold, a four-disulfide globular core from which emerge three loops or fingers and a C-terminal tail. An NMR dynamics study of a short α-neurotoxin reveals "disorder" at the tips of fingers I and II reflecting localized mobility on the picosecond to nanosecond time scales (9.Connolly P.J. Stern A.S. Hoch J.C. Biochemistry. 1996; 35: 418-426Crossref PubMed Scopus (19) Google Scholar). The significance of this mobility is highlighted by evidence that mutation of residues at these locations in other related toxins produces large effects on binding (11.Trémeau O. Lemaire C. Drevet P. Pinkasfeld S. Ducancel F. Boulain J.-C. Ménez A. J. Biol. Chem. 1995; 270: 9362-9369Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). α-Bungarotoxin (Bgtx), a long neurotoxin from the venom of Bungarus multicinctus, has been an important tool in many biochemical and functional studies of nAChRs including the homo-pentameric nAChRs. The three-dimensional structure of Bgtx, determined by x-ray crystallography (12.Love R.A. Stroud R.M. Protein Eng. 1986; 1: 37-46Crossref PubMed Scopus (156) Google Scholar), differed from solution and x-ray structures of related short and long toxins particularly in the length of the highly conserved central β-sheet in finger II (6.Zinn-Justin S. Roumestand C. Gilquin B. Bontems F. Ménez A. Toma F. Biochemistry. 1992; 31: 11335-11347Crossref PubMed Scopus (87) Google Scholar, 7.Leroy E. Mikou A. Yang Y. Guittet E. J. Biomol. Struct. Dyn. 1994; 12: 1-17Crossref PubMed Scopus (5) Google Scholar, 8.Betzel C. Lange G. Pal G.P. Wilson K.S. Maelicke A. Saenger W. J. Biol. Chem. 1991; 266: 21530-21536Abstract Full Text PDF PubMed Google Scholar). In contrast, an NMR-based investigation of the secondary structure in Bgtx indicated that the structure of Bgtx in solution most likely resembles that of other α-neurotoxins (13.Basus V.J. Billeter M. Love R.A. Stroud R.M. Kuntz I.D. Biochemistry. 1988; 27: 2763-2771Crossref PubMed Scopus (79) Google Scholar). Although this study paved the way to a better understanding of the solution structure of Bgtx, an adequately high resolution structure of Bgtx has not been available until now. The high resolution three-dimensional structure of unbound Bgtx presented here greatly facilitates the investigation of conformational changes that Bgtx undergoes as it binds nAChR-derived and engineered sequences, the subject of recent structural studies (14.Basus V.J. Song G. Hawrot E. Biochemistry. 1993; 32: 12290-12298Crossref PubMed Scopus (95) Google Scholar, 15.Gentile L.N. Basus V.J. Shi Q.-L. Hawrot E. Ann. N. Y. Acad. Sci. 1995; 757: 222-237Crossref PubMed Scopus (9) Google Scholar, 16.Zeng H. Moise L. Grant M.A. Hawrot E. J. Biol. Chem. 2001; 276: 22930-22940Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17.Scherf T. Balass M. Fuchs S. Katchalski-Katzir E. Anglister J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6059-6064Crossref PubMed Scopus (40) Google Scholar, 18.Scherf T. Kasher R. Balass M. Fridkin M. Fuchs S. Katchalski-Katzir E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6629-6634Crossref PubMed Scopus (61) Google Scholar, 19.Samson A.O. Chill J.H. Rodriguez E. Scherf T. Anglister J. Biochemistry. 2001; 40: 5464-5473Crossref PubMed Scopus (20) Google Scholar, 20.Harel M. Kasher R. Nicolas A. Guss J.M. Balass M. Fridkin M. Smit A.B. Brejc K. Sixma T.K. Katchalski-Katzir E. Sussman J.L. Fuchs S. Neuron. 2001; 32: 265-275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Photolabeling and mutagenesis studies indicate that the ligand-binding site on the nAChR is formed at subunit interfaces in the N-terminal extracellular region of the receptor (1.Karlin A. Akabas M.H. Neuron. 1995; 15: 1331-1344Abstract Full Text PDF Scopus (563) Google Scholar). The major structural determinants of ligand binding are found on the α subunit with additional contributions made by residues of adjoining subunits. Strongly conserved residues from three discontinuous regions of the α subunit (designated loops A–C) contribute to the binding pocket. In the crystal structure of the acetylcholine-binding protein (AChBP), a snail homologue of the extracellular domain of a homo-pentameric nAChR, loops A–C contribute to a relatively hydrophobic cavity at the subunit interface (21.Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1574) Google Scholar). Earlier studies (22.Wilson P.T. Lentz T.L. Hawrot E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8790-8794Crossref PubMed Scopus (121) Google Scholar) have shown that the major determinants of Bgtx binding lie between positions 173 and 204 on the α1 subunit, coincident with loop C. This region includes the highly conserved aromatic residues Tyr190 and Tyr198and, in addition, Cys192 and Cys193 (in α1 numbering) which form an uncommon disulfide. Synthetic peptide binding studies have suggested that the Bgtx-binding site on the α7 subunit is in the homologous region (α7 178–196) (23.McLane K.E. Wu X. Schoepfer R. Lindstrom J.M. Conti-Tronconi B.M. J. Biol. Chem. 1991; 266: 15230-15239Abstract Full Text PDF PubMed Google Scholar). The importance of this region in toxin binding was further highlighted by the observation that six residues from this region of α7 could confer Bgtx sensitivity when placed into the corresponding positions of the Bgtx-insensitive α3 subunit (24.Levandoski M.M. Lin Y. Moise L. McLaughlin J.T. Cooper E. Hawrot E. J. Biol. Chem. 1999; 274: 26113-26119Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Additionally, mutation of loop C residues, Tyr187 and Tyr194 of α7, to Phe reduced Bgtx blockade of ACh-evoked currents in heterologously expressed receptors (25.Galzi J.-L. Bertrand D. Devillers-Thiéry A. Revah F. Bertrand S. Changeux J.P. FEBS Lett. 1991; 294: 198-202Crossref PubMed Scopus (137) Google Scholar). This is consistent with a 60-fold reduction in Bgtx binding observed in synthetic peptides where Tyr190of α1 (which corresponds to Tyr187 of α7) was mutated to Phe, showing the important role of Tyr190 in binding Bgtx (26.Pearce S.F.A. Preston-Hurlburt P. Hawrot E. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1990; 241: 207-213Crossref PubMed Scopus (20) Google Scholar). Previously, we reported NMR solution structures for two α1 subunit-derived peptides, an α12-mer (α185–196) and an α18-mer (α181–198), in complex with Bgtx (14.Basus V.J. Song G. Hawrot E. Biochemistry. 1993; 32: 12290-12298Crossref PubMed Scopus (95) Google Scholar, 15.Gentile L.N. Basus V.J. Shi Q.-L. Hawrot E. Ann. N. Y. Acad. Sci. 1995; 757: 222-237Crossref PubMed Scopus (9) Google Scholar, 16.Zeng H. Moise L. Grant M.A. Hawrot E. J. Biol. Chem. 2001; 276: 22930-22940Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Here we extend our analysis of the Bgtx-binding site to the corresponding 19-amino acid segment on the neuronal nAChR α7 subunit (α7 178–196) with an original solution NMR structure of the α7 19-mer in complex with Bgtx. Our primary goals are to determine an energetically favorable conformation for a region of the neuronal nAChR important in agonist and antagonist binding and to understand the structural basis for the strong interaction between Bgtx and the α7 nAChR. In addition, we present a new high resolution analysis of the secondary structure-rich core of Bgtx, significantly extending the initial NMR structural studies of this toxin (13.Basus V.J. Billeter M. Love R.A. Stroud R.M. Kuntz I.D. Biochemistry. 1988; 27: 2763-2771Crossref PubMed Scopus (79) Google Scholar). We prepared a synthetic gene encoding residues 178–196 (IPGKRTESFYECCKEPYPD) of the chick neuronal nAChR α7 subunit (27.Courtier S. Bertrand D. Matter J.M. Hernandez M.C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (816) Google Scholar) using mutually priming oligonucleotides. The oligonucleotides were designed according to the specifications of the pET-31 Peptide Expression System (Novagen) with 3′ overhangs encoding methionine (28.Kuliopulos A. Walsh C.T. J. Am. Chem. Soc. 1994; 116: 4599-4607Crossref Scopus (123) Google Scholar). The sequences of the two oligonucleotides are 5′-ATTCCGGGCAAACGTACCGAAAGCTTCTATGAATGCTGCAAAGAACCGTATCCGGATATG-3′ and 5′-ATCCGGATACGGTTCTTTGCAGCATTCATAGAAGCTTTCGGTACGTTTGCCCGGAATCAT-3′. Two copies of this expression cassette were ligated in tandem into pET-31b(+), and the construct was authenticated by DNA sequencing in the forward and reverse directions. The α7 19-mer was expressed as a ketosteroid isomerase fusion protein with a C-terminal polyhistidine tag in Escherichia coli BL21(DE3)pLysS cells (Novagen). Isotopically labeled α7 19-mer was produced in E. coli using M9 minimal medium with (15NH4)2SO4 and 13C6-glucose (Cambridge Isotope Laboratories) as the sole sources of nitrogen and carbon, respectively (29.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: A.3Google Scholar). The medium was supplemented with vitamins according to Ref. 30.Venters R.A. Farmer B.T. Fierke C.A. Spicer L.D. J. Mol. Biol. 1996; 264: 1101-1116Crossref PubMed Scopus (173) Google Scholar. Detailed methods for expression and purification of the α7 19-mer have been reported previously (16.Zeng H. Moise L. Grant M.A. Hawrot E. J. Biol. Chem. 2001; 276: 22930-22940Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) for a similar nAChR recombinant peptide. Briefly, ketosteroid isomerase-α7 19-mer fusion protein was isolated from resolubilized inclusion bodies by nickel affinity chromatography. The fusion protein was then treated with CNBr and the reaction products separated by RP-HPLC. α7 19-mer was isocratically eluted from a semi-preparative C18RP-HPLC column (Vydac) at 5 ml/min in 20% acetonitrile, 0.1% trifluoroacetic acid. Mass spectrometric analysis of HPLC fractions identified purified α7 19-mer (HHMI Biopolymer/Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine). The redox status of the adjacent cysteine residues, Cys189–Cys190, was determined by RP-HPLC analysis of N-ethylmaleimide-treated α7 19-mer in its condition after purification and following pretreatment with dithiothreitol. Typical yields of isotopically labeled α7 19-mer were 2–4 mg/liter. Purified peptide was lyophilized and stored at −20 °C. The KD value for the α7 19-mer-Bgtx interaction was determined by measurement of inhibition of the initial rate of Bgtx binding to nAChR-enriched Torpedo membranes following preincubation of Bgtx with α7 19-mer (26.Pearce S.F.A. Preston-Hurlburt P. Hawrot E. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1990; 241: 207-213Crossref PubMed Scopus (20) Google Scholar). 125I-Labeled Bgtx (2.5 nm) was incubated over a wide range of concentrations of α7 19-mer in 0.2% bovine serum albumin, 30 mm sodium phosphate, pH 7.4, for 18 h at room temperature. 100 μl of the peptide/toxin mixture was added to microtiter plate wells coated with 2 μg of Torpedo membranes that were pre-blocked with 200 μl of 2% bovine serum albumin for 1 h. Following a 5-min incubation, the binding reaction was aspirated, and wells were washed 4 times with 200 μl of 0.2% bovine serum albumin, 30 mm sodium phosphate, pH 7.4. Torpedo membrane-bound 125I-Bgtx was measured in a γ counter. All measurements were done in triplicate. The effect of pH was also assessed by preparing samples in 30 mm sodium phosphate, pH 5.5, to replicate the buffer conditions of the NMR sample (see below). Hydrated Bgtx (Sigma) was used to resuspend the α7 19-mer in order to facilitate formation of a 1:1 peptide-toxin complex. Uniformly 15N-labeled and uniformly15N,13C-double-labeled α7 19-mer·Bgtx samples were prepared at a concentration of 1.5–2.0 mm. These samples contained 30 mm sodium phosphate and 50 μm sodium azide in 95% H2O, 5% D2O at pH 5.5. Sodium 3-trimethylsilylpropionate (Cambridge Isotope Laboratories) was added at 50 μm as an internal calibration standard. For deuterium exchange experiments, the complex was lyophilized and reconstituted in 99.9% D2O at pH 5.5 (isotope effect unaccounted). Preparation of the free Bgtx samples was described previously (13.Basus V.J. Billeter M. Love R.A. Stroud R.M. Kuntz I.D. Biochemistry. 1988; 27: 2763-2771Crossref PubMed Scopus (79) Google Scholar). NMR experiments were carried out at1H frequencies of 400 and 600 MHz on Bruker Avance spectrometers at Brown University and at 500 MHz on a GE spectrometer equipped either with a GN console with a Nicolet computer or an Omega console with a Sun 3/160 computer at the University of California, San Francisco. For free Bgtx, the following spectra in H2O were acquired at 15, 25, and 35 °C, and pH 5.79: DQF-COSY (31.Rance M. Sørensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2596) Google Scholar), TOCSY (70 ms MLEV-17 spin-lock sequence) (32.Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3104) Google Scholar), and NOESY spectra (160-ms mixing time) (33.Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4828) Google Scholar) using the water suppression scheme described in Basus (34.Basus V.J. J. Magn. Reson. 1984; 60: 138-142Google Scholar). No significant shifts in free Bgtx proton resonances were observed between samples at pH 5.5 and 5.79, suggesting that the chemical environment is comparable at both pH conditions. In addition, NOESY (160-ms mixing time) and E-COSY spectra (35.Griesinger C. Sørensen O.W. Ernst R.R. J. Am. Chem. Soc. 1985; 107: 6394-6396Crossref Scopus (517) Google Scholar) at 25 and 35 °C in D2O were acquired. Distance restraints were calculated from experimental NOESY intensities using the program MARDIGRAS version 2.0 (36.Borgias B.A. James T.L. Methods Enzymol. 1989; 176: 169-183Crossref PubMed Scopus (133) Google Scholar, 37.Borgias B.A. James T.L. J. Magn. Reson. 1990; 87: 475-487Google Scholar) which uses the complete relaxation matrix to produce an upper and lower distance bound for each experimental intensity. To make use of this procedure that determines accurate distances from the integrated intensities of the NOESY cross-peaks, a value for the rotational correlation time, τc, must be defined. For free Bgtx the correlation time was determined by measurement of the 13C T1 and T2relaxation times at natural abundance, using a sample dissolved in 99.96% D2O. These experiments were carried out using a double-DEPT technique with proton detection for maximum sensitivity. For T1, we used the double-DEPT sequence with inversion recovery (38.Sklenár V. Torchia D. Bax A. J. Magn. Reson. 1987; 73: 375-379Google Scholar), and for T2, we used the double-DEPT sequence with a Carr-Purcell-Meiboom-Gill modification using a series of 180° pulses with a repetition rate of 1 ms to replace the single 180° refocusing pulse in the sequence of Nirmala and Wagner (39.Nirmala N.R. Wagner G. J. Magn. Reson. 1989; 82: 659-661Google Scholar). The sum of the resonances at different portions of the spectra was used to determine the relaxation times. The data were analyzed by fitting to a single exponential decay function. Peak volumes from D2O and H2O NOESY spectra were obtained by fitting the peak or peaks to be integrated to a Gaussian line shape in Sparky. In the case of peaks with low signal to noise ratio, the points within a manually selected rectangular or elliptical area surrounding the cross-peak were summed using Sparky (40.Goddard T.D. Kneller D.G. SPARKY 3. University of California, San Francisco, CA2001Google Scholar). In NOESY spectra the 3 JHNα coupling constants were determined by line fitting as described above, using Sparky. These values were used as the minimum values, because the apparent coupling constant in NOESY spectra will be smaller than the actual coupling constant. In DQF-COSY spectra, coupling constants were determined by line fitting the antiphase multiplets, followed by measurement of the separation between the simulated anti-phase multiplets (35.Griesinger C. Sørensen O.W. Ernst R.R. J. Am. Chem. Soc. 1985; 107: 6394-6396Crossref Scopus (517) Google Scholar). These values were used as maximum values, because the apparent coupling in DQF-COSY spectra is larger than the actual coupling constant due to the summation of anti-phase cross-peaks. Exchange rates were measured at 25 °C for Bgtx lyophilized from H2O and re-dissolved in D2O. Immediately following this procedure, the sample was placed in the spectrometer, and one-dimensional and TOCSY spectra (40 ms mixing time) were alternately acquired. The first one-dimensional spectrum was acquired 14 min after dissolving the lyophilized powder in D2O, and the first TOCSY spectrum was started 1 min later. Several spectra were obtained up to 36 h after starting the exchange, and further spectra were obtained 1 and 2 weeks later with the sample maintained at 25 °C. The cross-peaks in the resulting spectra were integrated, and cross-peaks between α- and β-protons were integrated for use as intensity references to eliminate variations of the spectrometer conditions after the sample was removed and re-inserted in the magnet for the last time points. For the α7 19-mer·Bgtx complex, 1H-15N three-dimensional NOESY-HSQC (120-ms mixing time) (41.Marion D. Driscoll P.C. Kay L.E. Wingfield P.T. Bax A. Gronenborn A.M. Clore G.M. Biochemistry. 1989; 28: 6150-6156Crossref PubMed Scopus (932) Google Scholar),1H-15N three-dimensional TOCSY-HSQC (60-ms MLEV-17 spin-lock sequence) (41.Marion D. Driscoll P.C. Kay L.E. Wingfield P.T. Bax A. Gronenborn A.M. Clore G.M. Biochemistry. 1989; 28: 6150-6156Crossref PubMed Scopus (932) Google Scholar), and three-dimensional HNHA (42.Vuister G.W. Bax A. J. Am. Chem. Soc. 1993; 115: 7772-7777Crossref Scopus (1048) Google Scholar) experiments were collected at 35 °C using a uniformly15N-labeled α7 19-mer·Bgtx sample in order to assign resonances. To clarify ambiguities in the assignments, an HNCA experiment (43.Grzesiek S. Bax A. J. Magn. Reson. 1992; 96: 432-440Google Scholar) was performed using uniformly15N/13C double-labeled α7 19-mer.1H homonuclear NOESY (120-ms mixing time) (33.Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4828) Google Scholar) and TOCSY (60-ms MLEV-17 spin-lock sequence) (32.Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3104) Google Scholar) experiments were performed with15N decoupling to assign bound Bgtx resonances. NOESY and TOCSY experiments were performed at 15, 25, and 35 °C and with different mixing times to resolve ambiguities and facilitate the assignment process. Water was suppressed using the WATERGATE method, incorporating a 3-9-19 refocusing pulse sequence with pulsed field gradients (44.Sklenár V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. Ser. A. 1993; 102: 241-245Crossref Scopus (1109) Google Scholar). Deuterium exchange was performed to identify slowly exchanging amide protons involved in secondary structure. Following a two-dimensional 1H-15N HSQC (45.Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-189Crossref Scopus (2417) Google Scholar), five (sequential) homonuclear TOCSY spectra were collected over 16 h following reconstitution of the α7 19-mer·Bgtx complex in D2O. The stoichiometry of the α7 19-mer·Bgtx complex was determined in a mole ratio titration of the α7 19-mer using two-dimensional 1H-15N HSQC experiments. NMR data were processed by XWIN-NMR (Bruker) or NMRPipe (46.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11450) Google Scholar), and resonance assignments were made in Sparky (40.Goddard T.D. Kneller D.G. SPARKY 3. University of California, San Francisco, CA2001Google Scholar). 1H chemical shifts were referenced to sodium 3-trimethylsilylpropionate (0.0 ppm). The1H, 15N, and 13C assignments will be deposited in the BioMagResBank chemical shift data base. For free Bgtx, distance geometry calculations were performed on the Cray Y-MP at the San Diego Supercomputer Center using the distance geometry program VEMBED (47.Kuntz I.D. Thomason J.F. Oshiro C.M. 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