Advertisement

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 *

  • Haoyu Zeng
    Footnotes
    Affiliations
    ‡Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown Medical School, Providence, Rhode Island 02912
    Search for articles by this author
  • Leonard Moise
    Affiliations
    ‡Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown Medical School, Providence, Rhode Island 02912
    Search for articles by this author
  • Marianne A. Grant
    Footnotes
    Affiliations
    ‡Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown Medical School, Providence, Rhode Island 02912
    Search for articles by this author
  • Edward Hawrot
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    ‡Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown Medical School, Providence, Rhode Island 02912
    Search for articles by this author
  • Author Footnotes
    * This research was supported by National Institutes of Health Research Grants GM32629 and NS34348 (to E. H.). NMR instrumentation was funded by National Institutes of Health Grant RR08240 and National Science Foundation Grant DBI-9723282.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The atomic coordinates and the structure factors (code , , , and ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
    § This work was done in partial fulfillment of the requirements for a Ph.D. degree from Brown University.
    ¶ Present address: Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Research East 319, 41 Ave. Louis Pasteur, Boston, Massachusetts 02215.
      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.
      nAChR
      nicotinic acetylcholine receptor
      Bgtx
      α-bungarotoxin
      HPLC
      high performance liquid chromatography
      CNBr
      cyanogen bromide
      HSQC
      heteronuclear single quantum correlation
      TOCSY
      total correlation spectroscopy
      NOE
      nuclear Overhauser effect
      NOESY
      nuclear Overhauser enhancement spectroscopy
      r.m.s.d.
      root mean square deviation
      NmmI
      Naja mossambica mossambica I
      The nicotinic acetylcholine receptor (nAChR)1 (
      • Karlin A.
      • Akabas M.H.
      ) 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.
      • Karlin A.
      • Akabas M.H.
      ).
      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 (
      • Antil S.
      • Servent D.
      • Ménez A.
      ,
      • Malany S.
      • Osaka H.
      • Sine S.M.
      • Taylor P.
      ,
      • Rosenthal J.A.
      • Levandoski M.M.
      • Chang B.
      • Potts J.F.
      • Shi Q.-L.
      • Hawrot E.
      ,
      • Rosenthal J.A.
      • Hsu S.H.
      • Schneider D.
      • Gentile L.N.
      • Messier N.J.
      • Vaslet C.A.
      • Hawrot E.
      ).
      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 (
      • Love R.A.
      • Stroud R.M.
      ). Previous NMR structural studies indicate that the solution structure of Bgtx, although generally consistent with the x-ray structure, does reveal some differences (
      • Basus V.J.
      • Billeter M.
      • Love R.A.
      • Stroud R.M.
      • Kuntz I.D.
      ). 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 (
      • Love R.A.
      • Stroud R.M.
      ). The structures of several other snake venom α-neurotoxins have been studied with NMR techniques (
      • Labhardt A.M.
      • Hunziker-Kwik E.H.
      • Wüthrich K.
      ,
      • Le-Goas R.
      • LaPlante S.R.
      • Mikou A.
      • Delsuc M.A.
      • Guittet E.
      • Robin M.
      • Charpentier I.
      • Lallemand J.Y.
      ,
      • Zinn-Justin S.
      • Roumestand C.
      • Gilquin B.
      • Bontems F.
      • Ménez A.
      • Toma F.
      ,
      • Connolly P.J.
      • Stern A.S.
      • Hoch J.C.
      ,
      • Peng S.-S.
      • Kumar T.K.S.
      • Jayaraman G.
      • Chang C.-C.
      • Yu C.
      ), 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 (
      • Wilson P.T.
      • Lentz T.L.
      • Hawrot E.
      ), a region that coincides with one of three segments of the α subunit that have been implicated in agonist binding (for review, see Ref.
      • Karlin A.
      • Akabas M.H.
      ). Tyr190, along with Cys192, Cys193, and Tyr198 are selectively cross-linked with a variety of site-directed photoaffinity reagents (
      • Grutter T.
      • Ehret-Sabatier L.
      • Kotzyba-Hibert F.
      • Goeldner M.
      ). 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 (
      • Kachalsky S.G.
      • Jensen B.S.
      • Barchan D.
      • Fuchs S.
      ,
      • Wilson P.T.
      • Hawrot E.
      • Lentz T.L.
      ). 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 (
      • Pearce S.F.A.
      • Preston-Hurlburt P.
      • Hawrot E.
      ). 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 (
      • Pearce S.F.A.
      • Preston-Hurlburt P.
      • Hawrot E.
      ). Mutations of Tyr190, when assessed in heterologous expression systems, also result in large decreases in α-neurotoxin binding (
      • Malany S.
      • Osaka H.
      • Sine S.M.
      • Taylor P.
      ,
      • Spura A.
      • Riel R.U.
      • Freedman N.D.
      • Agrawal S.
      • Seto C.
      • Hawrot E.
      ,
      • Ackermann E.J.
      • Ang E.T.-H.
      • Kanter J.R.
      • Tsigelny I.
      • Taylor P.
      ). In addition, ligand gating is also dramatically affected by mutations in Tyr190 (
      • Tomaselli G.F.
      • McLaughlin J.T.
      • Jurman M.E.
      • Hawrot E.
      • Yellen G.
      ,
      • Galzi J.L.
      • Bertrand D.
      • Devillers-Thiery A.
      • Revah F.
      • Bertrand S.
      • Changeux J.P.
      ). 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 (
      • Kachalsky S.G.
      • Jensen B.S.
      • Barchan D.
      • Fuchs S.
      ).
      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 (
      • Pearce S.F.A.
      • Preston-Hurlburt P.
      • Hawrot E.
      ,
      • Basus V.J.
      • Song G.
      • Hawrot E.
      ). 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.

      EXPERIMENTAL PROCEDURES

       Expression Construct

      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(+) (
      • Kuliopulos A.
      • Walsh C.T.
      ). 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 Growth

      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.

       Nickel Affinity Chromatography

      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)).

       CNBr Cleavage

      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.

       Reverse Phase-HPLC Purification

      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.

       NMR Sample Preparation

      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.

       NMR Experiments

      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) (
      • Piotto M.
      • Saudek V.
      • Sklenar V.
      ,
      • Sklenar V.
      • Piotto M.
      • Leppik R.
      • Saudek V.
      ,
      • Bodenhausen G.
      • Ruben D.J.
      ) experiment. Amino acid spin systems were identified by two-dimensional total correlation spectroscopy (TOCSY) (
      • Piotto M.
      • Saudek V.
      • Sklenar V.
      ,
      • Sklenar V.
      • Piotto M.
      • Leppik R.
      • Saudek V.
      ,
      • Bax A.
      • Davis D.G.
      ) and three-dimensional TOCSY-HSQC experiments (
      • Bax A.
      • Davis D.G.
      ,
      • Davis A.L.
      • Keeler J.
      • Laue E.D.
      • Moskau D.
      ,
      • Palmer III A.G.
      • Cavanagh J.
      • Wright P.E.
      • Rance M.
      ,
      • Kay L.E.
      • Keifer P.
      • Saarinen T.
      ,
      • Schleucher J.
      • Sattler M.
      • Griesinger C.
      ) 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 (
      • Vuister G.W.
      • Bax A.
      ,
      • Vuister G.W.
      • Bax A.
      ). 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) (
      • Piotto M.
      • Saudek V.
      • Sklenar V.
      ,
      • Sklenar V.
      • Piotto M.
      • Leppik R.
      • Saudek V.
      ) and three-dimensional NOESY-HSQC experiments (
      • Davis A.L.
      • Keeler J.
      • Laue E.D.
      • Moskau D.
      ,
      • Palmer III A.G.
      • Cavanagh J.
      • Wright P.E.
      • Rance M.
      ,
      • Kay L.E.
      • Keifer P.
      • Saarinen T.
      ,
      • Schleucher J.
      • Sattler M.
      • Griesinger C.
      ) 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 (
      • Delaglio F.
      • Grzesiek S.
      • Vuister G.W.
      • Zhu G.
      • Pfeifer J.
      • Bax A.
      ), and SPARKY (
      • Goddard T.D.
      • Kneller D.G.
      ).
      In comparing our results with earlier, more preliminary assignments involving an unlabeled α18-mer peptide bound to Bgtx (
      • Gentile L.N.
      • Basus V.J.
      • Shi Q.-L.
      • Hawrot E.
      ), 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 (
      • Gentile L.N.
      • Basus V.J.
      • Shi Q.-L.
      • Hawrot E.
      ). The new swapped assignments incorporate the results from the three-dimensional HNHA experiment.

       Conformation Calculations

      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 (
      • Bax A.
      • Vuister G.W.
      • Grzesiek S.
      • Delaglio F.
      • Wang A.C.
      • Tschudin R.
      • Zhu G.
      ). The 3 Jcoupling constants were converted to dihedral angle restraints using previously described methods (
      • Thompson G.S.
      • Leung Y.-C.
      • Ferguson S.J.
      • Radford S.E.
      • Redfield C.
      ). 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 (
      • Brunger 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.
      ). 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 (
      • Rölz C.
      • Mierke D.F.
      ). 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.
      Figure thumbnail gr7
      Figure 7Stereo views comparing the polypeptide backbone traces of bound and free Bgtx. A andC, a view of the ensemble of structures for free Bgtx and for the Bgtx·α18-mer complex. B and D, a view of the two mean structures calculated from the ensembles. InA and B, the views correspond to the concave surface of Bgtx. In C and D, Bgtx is rotated ∼90° to the right as compared with the view in A andB (the α18-mer is not shown). The red tracescorrespond to free Bgtx, the blue traces correspond to Bgtx in the Bgtx·α18-mer complex, and the green tracescorrespond to the α18-mer. The figures were prepared using the program MOLMOL (
      • Koradi R.
      • Billeter M.
      • Wüthrich K.
      ).
      We used Rasmol (
      • Sayle R.A.
      • Milner-White E.J.
      ), MOLMOL (
      • Koradi R.
      • Billeter M.
      • Wüthrich K.
      ), 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 (
      • Sobolev V.
      • Sorokine A.
      • Prilusky J.
      • Abola E.E.
      • Edelman M.
      ). The energetically significant cation-π interaction analysis of the Bgtx·α18-mer complex structures was performed using the CaPTURE program (
      • Gallivan J.P.
      • Dougherty D.A.
      ).

      RESULTS

       Preparation of 15N-peptide and Its Purification

      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.
      Figure thumbnail gr1
      Figure 1C18-reverse phase HPLC purification of the α18-mer. The peptide peak labeled P1 corresponds to the disulfide homoserine form of the α18-mer; P2 is the disulfide homoserine lactone form of the α18-mer that was collected and used in this NMR study. P4 is an unidentified form of the α18-mer, which slowly converts to P2. See “Experimental Procedures” for a detailed description of chromatographic conditions.

       The Formation of a Stoichiometric 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.
      Figure thumbnail gr2
      Figure 2Comparison of the HSQC spectra of free α18-mer and of the Bgtx·α18-mer complex. A, HSQC spectrum of free α18-mer at a concentration of 1.0 mm.B, HSQC spectrum of the stoichiometric Bgtx·α18-mer complex at a concentration of 2.1 mm. The NH resonance assignments were determined as described in the text. W184 S. C. is the side chain NH of Trp184; W187 S. C. is the side chain NH of Trp187.

       NMR Assignments

      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-dimensional NMR data obtained with the complex.
      Figure thumbnail gr3
      Figure 3Multi-dimensional NMR strip analysis of the 15N-enriched α18-mer illustrating the assignment of Lys185. Strip A is from a three-dimensional TOCSY-HSQC experiment that identifies the amino acid spin system of Lys185. Strip B is from a three-dimensional NOESY-HSQC experiment that depicts the short and medium range NOEs involving the NH of Lys185, and strip C is from a three-dimensional HNHA experiment showing the correlation between the NH proton and the CαH proton of Lys185. NH, amide proton;dαH, the sequential CαH proton from Trp184; αH, the CαH proton; dβH, the sequential CβH proton from Trp184; βHs, the CβH protons.
      Based on our three-dimensional NMR assignments, our two-dimensional NMR data (NOESY and TOCSY), and published Bgtx assignments (
      • Basus V.J.
      • Billeter M.
      • Love R.A.
      • Stroud R.M.
      • Kuntz I.D.
      ,
      • Gentile L.N.
      • Basus V.J.
      • Shi Q.-L.
      • Hawrot E.
      ), we completed the assignment of the resonances obtained with the Bgtx·α18-mer complex. Fig. 4summarizes the CαHi proton to NHi+1 proton NOEs (sequential NOE), the CβHi proton to NHi+1 proton NOEs, and the NHi proton to NHi+1proton NOEs in the Bgtx·α18-mer complex used to complete the connectivity of the polypeptide backbone. In addition, 11 unambiguous intermolecular NOEs on the NH region of the peptide were assigned (Table I). Additional intermolecular NOEs in the CβH proton and CγH proton regions were observed but have not as yet been unambiguously assigned. We performed a similar analysis on free Bgtx to generate the structure important for comparison of the free and bound forms of Bgtx.
      Figure thumbnail gr4
      Figure 4Structurally relevant NOEs from the two-dimensional NOESY spectra of the Bgtx·α18-mer complex. The thickness of the bars indicates the NOE intensity (strong, d < 3.0 Å; medium, d < 4.0 Å; weak, d < 5.0 Å).
      Table IObserved intermolecular NOEs
      Bgtxα18-mer
      Thr8CβHCys192NH
      Pro10CγH1
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Asp195NH
      Pro10CγH2
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Asp195NH
      Pro10CδH
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Thr196NH
      Lys38CαHThr191NH
      Val39CαHTyr189CβH1
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Val39CγH2
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Tyr189NH
      Val40CγH
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Thr191NH
      Val40NHTyr190CαH
      Pro69CγH1
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Cys192NH
      Pro69CγH2
      These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”
      Cys192NH
      1-a These positions have not been stereospecifically assigned. Distance constraints were determined using a pseudoatom approach as described under “Experimental Procedures.”

       Three-dimensional Structure Calculations and Comparison

      The distance constraints resulting from the NOEs and the dihedral angle restraints obtained from the HN-Hα3 J couplings (TableII) were then incorporated into CNSsolve for structure determination. These calculations utilized both distance geometry and simulated annealing protocols. Twenty structures of free Bgtx and of the Bgtx·α18-mer complex with the lowest potential energy and no NOE violation larger than 0.5 Å are superimposed and shown as an ensemble in Fig. 5. In Fig.6, the mean structure of each ensemble, partially energy-minimized, is shown to illustrate some of the key structural features. The overall backbone atomic root mean square deviation (r.m.s.d.) between the individual structures and the mean structure of free Bgtx is 2.03 Å, whereas that of the Bgtx·α18-mer complex is 2.07 Å. The pairwise r.m.s.d. between the two mean structures is 6.05 Å.
      Table IIStructural statistics for free Bgtx and for the Bgtx · α18-mer complex
      Bgtx · α18-mer complexFree Bgtx
      Total number of distance constraints530457
      Intraresidue303289
      Sequential(‖i−j‖= 1)150116
      Medium range(1<‖i−j‖≤4)1112
      Long range(‖i−j‖>4)5540
      Intermolecular11
      α18-mer dihedral angle restraints9
      Distance constraint violations>0.5Å00
      Bgtx·α18-mer complex: overall r.m.s.d. to mean
      Backbone heavy atoms (Å)2.07
      All heavy atoms (Å)3.29
      Bgtx:overall r.m.s.d.to the mean structure
      Backbone heavy atoms(Å)(residues 1–72)1.811.98
      Backbone heavy atoms(Å)(residues 1–74)1.952.03
      All heavy atoms(Å)(residues 1–72)2.983.21
      All heavy atoms(Å)(residues 1–74)3.033.23
      Backbone r.m.s.d.for different regions
      Residues1–16(Å)(loop I)1.441.71
      Residues 17–21(Å)0.990.73
      Residues 22–28(Å)(stem I of loop II)0.810.75
      Residues 29–36(Å)(tip of loop II)1.291.14
      Residues 37–44(Å)(stem II of loop II)0.901.19
      Residues 45–60(Å)(loop III)1.441.53
      Residues 61–72(Å)(C-terminal tail)1.321.49
      Residues 181–198(Å)(α18-mer)2.19
      Figure thumbnail gr5
      Figure 5Structural comparison between free-Bgtx and the Bgtx·α18-mer complex. A, twenty superimposed structures calculated based on distance constraints in free Bgtx. The red lines mark those Bgtx regions that undergo large chemical shifts upon α18-mer binding.B, twenty superimposed structures calculated based on distance constraints for the Bgtx·α18-mer complex. The blue lines correspond to Bgtx, and the green lines signify the α18-mer. The black lines and the labels Nand C mark the N and C termini of Bgtx. The labelsN′ and C′ mark the N and C termini of the α18-mer. Only backbone traces are shown. The figures were prepared using the program MOLMOL (
      • Koradi R.
      • Billeter M.
      • Wüthrich K.
      ).
      Figure thumbnail gr6
      Figure 6Stereo views of the mean calculated structures for free Bgtx and for the Bgtx·α18-mer complex. A, free Bgtx. B, the Bgtx·α18-mer complex. These are mean structures calculated from the appropriate ensemble of the 20 lowest energy structures (see Fig. ). The structures have been partially energy minimized to remove artifacts from averaging.
      We did not find any inter-residue NOE constraints involving the last two residues of Bgtx, Pro73 and Gly74. If these two residues are omitted from the r.m.s.d. calculation for Bgtx, the overall backbone r.m.s.d. is 1.98 Å for free Bgtx and 1.81 Å for bound Bgtx in the Bgtx·α18-mer complex. A comparison of the r.m.s.d. determinations for various sequence segments within Bgtx indicates considerable regional variation in r.m.s.d. as summarized in Table II. The stem region of loop II is well defined (r.m.s.d. no greater than 0.90 Å in Bgtx·α18-mer and 1.19 Å in free Bgtx) compared with loop II when considered in its entirety (r.m.s.d. of 1.56 Å in Bgtx·α18-mer complex and 1.51 Å in free Bgtx). The tip of loop II can also be reasonably well superimposed (r.m.s.d. of 1.29 Å in Bgtx·α18-mer complex or 1.14 Å in free Bgtx).
      A comparison of free and bound Bgtx reveals that the proton resonances of a number of amino acids in Bgtx undergo large chemical shift changes upon peptide binding. These shift changes reflect significant alterations in the chemical environment of those protons. We find that Ala7 (CαH), Ser9(CαH, CβH), Ile11 (NH, CαH, CβH, CγH), Trp28 (NH), Cys29 (NH, CαH), Asp30 (NH), Phe32 (NH), Cys33(CβH), Arg36 (NH), Gly37 (NH, CαH), Lys38 (NH, CβH, CγH), Val39 (CβH), Val40 (NH, CβH), Lys52(CβH), Val57 (CβH), Lys64 (CγH), Asn66(CβH), Lys70 (NH, CαH), Gln71 (CβH), and Arg72(CαH, CβH, CγH) all are characterized by chemical shift changes greater than 0.2 ppm upon binding. These amino acid residues are highlighted in red in the backbone structures shown in Fig. 5 A. The changes in Ala7, Ser9, and Ile11 suggest that the outer tip of loop I participates in binding the α18-mer. This is substantiated by the intermolecular NOEs observed between the CγH and CδH protons of Pro10and the NH protons of Asp195 and Thr196 in the peptide (Table I). Similarly, the chemical shift changes of Arg36-Val40 are correlated with intermolecular NOEs involving Lys38, Val39, and Val40 in Bgtx loop II. In the C-terminal tail region, the chemical shift changes in Lys70-Arg72 accompany intermolecular NOEs between the CγH protons of Pro69 and the NH of Cys192 in the peptide (Table I). In contrast to these examples of shift changes correlated with intermolecular contacts, several other shift changes in Bgtx residues appear to be due to conformational changes involving possible reorientations about the central triple-stranded β-sheet common to all α-neurotoxins. We believe that the chemical shift changes in the Trp28-Asp30, Phe32, Cys33 region and in Val57 are caused by general movement about the β-strand involving loop II and the proximal portion of loop III. Such secondary effects could also explain the chemical shift change of the side-chain CβH proton in Lys52 of loop III. Finally the chemical shift changes involving the side chains of Lys64 and Asn66 at the beginning of the C-terminal tail may reflect a change in chemical environment due to the apparent relocation of the distal C-terminal region to make a contact with the α18-mer peptide bridging the gap between Bgtx loop I and loop II (Fig. 5).
      As viewed in Fig. 6 A, free Bgtx is oriented with loop I (blue) on the left, loop III on the right, and the tip of loop II (green) at the bottom. This view corresponds with the so-called “concave” surface of the α-neurotoxins (
      • Love R.A.
      • Stroud R.M.
      ,
      • Peng S.-S.
      • Kumar T.K.S.
      • Jayaraman G.
      • Chang C.-C.
      • Yu C.
      ). In the free form of Bgtx, the tip of loop II forms the lower rim of this concave surface, and the loop I and loop II of free Bgtx are well separated with no inter-loop NOE constraints. The distance between the N of Ser34 and the N of Pro10 in free Bgtx is ∼31 Å. Upon peptide binding, both loop I and loop II interact with peptide residues (see below), as revealed by intermolecular NOEs (Table I). As a consequence of binding, loop I and II move closer to each other; the average distance between the N of Ser34 and the N of Pro10 narrows to ∼24 Å. In addition, the tip of loop II switches to a convex conformation upon binding (Fig. 6 and Fig.7, A–D). The C-terminal tail region of free Bgtx is relatively unconstrained in free Bgtx but participates in peptide interaction in the bound state (see below). The rest of the structure appears to change little upon complex formation (Fig. 6 B). To provide a full comparison of binding-induced structural changes in Bgtx, we have superimposed the mean backbone structures (Fig. 7, B and D) and the full ensemble of structures (Fig. 7, A and C) as stereo images. The red traces correspond to the backbone of free Bgtx, whereas the blue traces refer to the backbone of bound Bgtx. The α18-mer backbone is colored green. In the front view (Fig. 7, A and B), loop I and loop II move toward each other and toward the peptide, whereas loop III is largely unaltered. In the left-profile view, the α18-mer has been removed for a better view of the changes in the Bgtx backbone (Fig. 7,C and D). The C-terminal tail is shifted to the right (toward the peptide) in the bound state. The tip of loop II moves left, highlighting the change in general orientation in this region from a concave to a convex surface.
      To evaluate the significance of the observed change in the conformation of Bgtx upon binding, we compared the mean pairwise backbone r.m.s.d. within the ensemble of free structures with the mean pairwise backbone r.m.s.d. across ensembles (i.e. each bound structure to each free Bgtx structure). Such a comparison allows us to determine if the two data sets, the ensembles of free and bound structures, represent significantly different structures. The mean pairwise backbone r.m.s.d. among the 20 free Bgtx structures is 2.94 ± 0.38 Å. Similarly, among the 20 bound Bgtx structures, the pairwise r.m.s.d. is 2.82 ± 0.25 Å. In contrast, the mean pairwise backbone r.m.s.d. between the 20 bound Bgtx structures and the 20 free Bgtx structures is 6.05 ± 0.37 Å. Thus, the mean pairwise r.m.s.d. across the two ensembles differs by more than 6 S.D. from the mean pairwise r.m.s.d. for the ensemble of free (or bound) Bgtx structures. This analysis indicates that the ensemble of bound structures is indeed significantly different from the ensemble of free structures.
      We observed five pairs of CαH-CαH long-range NOEs in the two-dimensional NMR study of the α18-mer complex. These included the following pairs of residues: Cys23-Cys44, Tyr24-Cys59, Arg25-Leu42, Lys26-Val57, and Met27-Val40. These NOEs were previously reported as evidence for an anti-parallel triple-stranded β-sheet in the solution structure of Bgtx (
      • Basus V.J.
      • Billeter M.
      • Love R.A.
      • Stroud R.M.
      • Kuntz I.D.
      ), and similar β-sheet defining, inter-strand NOEs have been observed in the solution structures of other α-neurotoxins (
      • Peng S.-S.
      • Kumar T.K.S.
      • Jayaraman G.
      • Chang C.-C.
      • Yu C.
      ).

       Binding Interactions

      The structures indicate that Bgtx interacts with the α18-mer at three sites. These are the tip of loop I, the C-terminal tail region (intermolecular NOEs are obtained with Thr8, Pro10, and Pro69), and loop II residues facing loop I including Val39, Val40, and Lys38. Although Loop III of some α-neurotoxins (
      • Malany S.
      • Osaka H.
      • Sine S.M.
      • Taylor P.
      ) has been reported to be involved in binding to the nAChR, we did not observe any direct intermolecular NOEs between the α18-mer and loop III of Bgtx. The chemical shifts of most residues in loop III change little upon binding of the α18-mer, consistent with a lack of involvement of Bgtx loop III in α18-mer binding.
      The α18-mer residues responsible for the intermolecular NOEs with the Bgtx sites are Tyr189, Tyr190, Thr191, Cys192, Asp195, and Thr196. The contact zone is ∼18 Å in length (from N of Tyr189 to N of Thr196), and the total van der Waals contact surface areas range from 500 to 690 Å2, with a mean of 580 Å2. It is noteworthy that there is a cluster of four positive charged Bgtx residues (Arg36, Lys38, Lys70, Arg72) near the contact zone. In close apposition in the complex are found peptide residues Trp187, Tyr189, Tyr190(Fig. 8). This arrangement suggests the possibility of cation-π interactions (
      • Gallivan J.P.
      • Dougherty D.A.
      ) contributing energetically to the formation of the Bgtx·α18-mer complex. A cation-π interaction analysis carried out on the 120 Bgtx·α18-mer-calculated structures revealed that a total of 24 individual structures, including two from the 20 best structures, have energetically significant cation-π interactions as determined by CaPTURE (
      • Gallivan J.P.
      • Dougherty D.A.
      ). In this analysis, cation-π interactions are selected as energetically significant if the electrostatic energy is <−2.0 kcal/mol or, alternatively, if the electrostatic energy is <−1.0 kcal/mol and the van der Waals interaction for the pair is <−1.0 kcal/mol (
      • Gallivan J.P.
      • Dougherty D.A.
      ). As shown in Table III, the following candidate cation-π pairings were observed: Lys38/Tyr190, Lys38/Tyr189, Lys38/Trp187, Arg36/Tyr189, Arg36/Trp187, and Lys70/Tyr190. The Lys38/Tyr190 pairing was observed in 12 of these 24 structures, and one of these structures is shown in Fig.9.
      Figure thumbnail gr8
      Figure 8Stereo view of the surface charge profile of the Bgtx·α18-mer complex. Surface charge potentials were calculated as described under “Experimental Procedures.” Blue regions show positive charge, andred regions show negative charge. See Fig. B for orientation. The figure was prepared using the program MOLMOL (
      • Koradi R.
      • Billeter M.
      • Wüthrich K.
      ).
      Table IIIObserved cation-π interaction pairs
      Cation-π interaction pairNumber of structures observed
      Lys38/Tyr19012
      Lys38/Trp1878
      Lys38/Tyr1893
      Arg36/Tyr1891
      Arg36/Trp1871
      Lys70/Tyr1891
      CaPTURE (
      • Gallivan J.P.
      • Dougherty D.A.
      ) was used to analyze all 120 acceptable structures of the Bgtx · α18-mer complex for cation-π interactions. Two of the 24 structures identified contained two cation-π interaction pairs. In both cases, the two pairs were Lys38/Tyr190 and Lys38/Trp187.
      Figure thumbnail gr9
      Figure 9Orientation of a suggested Tyr190-Lys38 cation-π interaction. The two side chains are taken from one of the 20 ensemble Bgtx·α18-mer structures depicted in Fig. .a, the distance between the NZ of Lys38 and the CE2 of Tyr190 is 5.49 Å; b, the distance between the NZ of Lys38 and the CE1 of Tyr190is 5.85 Å; c, the distance between the CG of Tyr190 and the NZ of Lys38 is 5.53 Å.

      DISCUSSION

      NMR studies of complexes formed between peptides and peptide binding domains have been very instrumental in many other systems in elucidating mechanisms of molecular recognition (
      • Feng S.
      • Chen J.K., Yu, H.
      • Simon J.A.
      • Schreiber S.L.
      ). In seeking to understand the structural basis for the relatively high affinity (KD ∼ 65 nm) binding observed between Bgtx and the α18-mer, we identified a number of NOE distance constraints that reveal important information about the interaction between the α18-mer and Bgtx and that also define conformational changes in Bgtx upon complex formation. These constraints have been used to generate the structural features presented in this study. The disc-like shape of Bgtx and of the complex limits the total number of long-distance NOEs available (NOEs between residues distant in primary sequence), and this in turn limits the final resolution of the structures obtained (Table II). This limitation is applicable to all members of the α-neurotoxin family (
      • Connolly P.J.
      • Stern A.S.
      • Hoch J.C.
      ,
      • Peng S.-S.
      • Kumar T.K.S.
      • Jayaraman G.
      • Chang C.-C.
      • Yu C.
      ). Finally, we compare the structural features of the binding between Bgtx and the cognate α18-mer to the biochemical- and mutagenesis-based observations made with the intact nAChR.
      One of our most striking observations is the binding-associated re-orientation in several segments of Bgtx that normally are not in contact with one another. Loop I and the C-terminal tail segment alter their configuration with respect to the main body of the protein, and the tip of loop II undergoes a change in its relative curvature and shape (Fig. 7). Together, these changes suggest a considerable coordinated reconfiguration of Bgtx upon interacting with receptor sequences. Such a reconfiguration is consistent with the extensive flexibility that has been noted in these and other protein toxins. It has been suggested that such flexibility may have evolved to serve important functional purposes (e.g. Ref.
      • Goldenberg D.P.
      • Koehn R.E.
      • Gilbert D.E.
      • Wagner G.
      ).
      We previously reported the solution structure of the complex formed between Bgtx and a dodecapeptide corresponding to amino acid residues 185–196 from the α subunit of the Torpedo nAChR (
      • Basus V.J.
      • Song G.
      • Hawrot E.
      ). The apparent affinity of Bgtx for the α12-mer is about 15–20-fold lower than for the α18-mer. In both structures, Tyr189 and Tyr190 lie in a similar position, close to Val39 and Val40 of Bgtx. In addition, in both structures the peptides are relatively elongated. There are significant differences, however, in the orientation of the two peptides relative to Bgtx. In the Bgtx·α12-mer complex, the polypeptide backbone of the five identified amino acid residues is between loop I and loop II, in a position roughly parallel to loop II. In the Bgtx·α18-mer complex, the peptide is in a more perpendicular orientation with respect to loop II and the tip of loop I (Fig. 6).
      The Bgtx·α12-mer complex may represent an intermediate stage in binding with the aromatics of the peptide, Tyr189 and Tyr190, forming a nucleation site for further interactions. Although the Bgtx·α18-mer complex is more energetically favored, the total contact surface areas are very similar, ∼ 600 Å2, for the two complexes. A library-derived peptide selected for its ability to bind Bgtx with high affinity also contains two adjacent Tyr residues, and these residues contribute the largest contact area in the complex (
      • Scherf T.
      • Balass M.
      • Fuchs S.
      • Katchalski-Katzir E.
      • Anglister J.
      ). Although this 13-residue library-derived peptide adopts a more globular or sphere-like conformation, it is also found localized to the cleft formed between loop I and loop II with close apposition of the C-terminal region (
      • Scherf T.
      • Balass M.
      • Fuchs S.
      • Katchalski-Katzir E.
      • Anglister J.
      ).
      In the Bgtx·α18-mer complex, the intermolecular NOEs between Pro10 and both Asp195 and Thr196and between Thr8 and Cys192 (Table I) demonstrate the involvement of Bgtx loop I in the formation of the peptide complex. This finding is consistent with mutagenesis studies of erabutoxin a, a short α-neurotoxin. Loop I mutations, S8T and Q10A, in erabutoxin a result in a very large reduction in binding affinity for the Torpedo nAChR (
      • Antil S.
      • Servent D.
      • Ménez A.
      ). Recently, a double-mutant cycle analysis involving the related short α-neurotoxin, Naja mossambica mossambica I (NmmI), and the mouse nAChR has revealed an interaction between Ser8 in NmmI and Tyr198 on the α subunit at the αγ site (
      • Malany S.
      • Osaka H.
      • Sine S.M.
      • Taylor P.
      ). In contrast, no evidence of an interaction was observed between Ser8 and Val188 in the NmmI study. In addition, Val188 can be energetically coupled to Arg33and Arg36 in NmmI (
      • Ackermann E.J.
      • Ang E.T.-H.
      • Kanter J.R.
      • Tsigelny I.
      • Taylor P.
      ). Based on sequence alignment, the corresponding Bgtx residues would be Arg36and Lys38, respectively. These observations are entirely consistent with the structure of the Bgtx·α18-mer complex; the tip of loop I is in closer proximity to the peptide residues C-terminal to Thr196 (Tyr198 is located at the extreme left in Fig. 6 B), whereas Val188 is removed from loop I and in close proximity to the loop II residue, Val39(Table I).
      Mutational analysis of loop I residues in the long α-neurotoxin, α-cobratoxin from Naja kaouthia venom, failed to detect a significant role for this region in binding to the TorpedonAChR (
      • Antil S.
      • Servent D.
      • Ménez A.
      ). Because the sequence of the loop I region differs greatly between Bgtx and this α-cobratoxin and because loop I is two residues longer in Bgtx (12 versus 10 between the corresponding Cys residues delimiting loop I), it is possible that these two toxins differ in this region in their mode of interaction with the nAChR. In addition, recent comparisons of short and long α-neurotoxins suggest significant differences between these two families of toxins in their detailed mode of interaction with the nAChR (
      • Antil S.
      • Servent D.
      • Ménez A.
      ,
      • Spura A.
      • Riel R.U.
      • Freedman N.D.
      • Agrawal S.
      • Seto C.
      • Hawrot E.
      ).
      The intermolecular NOE between Pro69 and the backbone NH of Cys192 together with the chemical shift changes observed within the C-terminal tail region upon formation of the Bgtx·α18-mer complex clearly indicate that the C-terminal tail region of Bgtx also plays a role in peptide binding. This observation is consistent with biochemical and mutagenesis studies examining the role of the C-terminal tail region in binding to native nAChRs where binding affinity was decreased by 7–15-fold when C-terminal residues were removed (
      • Rosenthal J.A.
      • Levandoski M.M.
      • Chang B.
      • Potts J.F.
      • Shi Q.-L.
      • Hawrot E.
      ,
      • Wu S.-H.
      • Chen C.-J.
      • Tseng M.-J.
      • Wang K.-T.
      ).
      The proximity of the positively charged Bgtx residues (Lys38 and Arg36) with the aromatics of the peptide (Tyr189 and Tyr190) in the Bgtx·α18-mer complex suggests an important functional contact. Mutagenesis studies in Bgtx and related toxins also point to important roles for Arg36 and Lys38. Ala-substitution of Arg36 in Bgtx leads to a 90-fold decrease in Bgtx binding affinity as measured with heterologously expressed mouse nAChR (
      • Rosenthal J.A.
      • Levandoski M.M.
      • Chang B.
      • Potts J.F.
      • Shi Q.-L.
      • Hawrot E.
      ), whereas charge reversal studies in α-cobratoxin demonstrate that R33E (position corresponds to Arg36 in Bgtx) causes a 767-fold decrease in binding affinity for Torpedo nAChR (
      • Antil S.
      • Servent D.
      • Ménez A.
      ). Ala-substitution at Arg36, the position corresponding to Lys38 in Bgtx, reduces binding affinity by 7.4-fold (
      • Antil S.
      • Servent D.
      • Ménez A.
      ).
      An important contact role for Tyr189 is consistent with chimeric analysis and toxin footprinting studies of nAChR α subunits. The α3 subunit, which shows no sensitivity to Bgtx block, acquires a significant sub-micromolar affinity for Bgtx with a single point mutation, K189Y, involving the introduction of an aromatic residue at position 189 (
      • Levandoski M.M.
      • Lin Y.
      • Moise L.
      • McLaughlin J.T.
      • Cooper E.
      • Hawrot E.
      ). In a footprinting protection study using Cys-substituted mutations in the heterologously expressed mouse α1 subunit, the introduced thiol of αF189C is protected by Bgtx from reaction with a hydrophilic biotinylmaleimide (
      • Spura A.
      • Riel R.U.
      • Freedman N.D.
      • Agrawal S.
      • Seto C.
      • Hawrot E.
      ). Similarly, the functional importance of Tyr190 in α-neurotoxin binding is supported by mutagenesis studies where large decreases in Bgtx andNmmI toxin binding affinity are observed (
      • Malany S.
      • Osaka H.
      • Sine S.M.
      • Taylor P.
      ,
      • Spura A.
      • Riel R.U.
      • Freedman N.D.
      • Agrawal S.
      • Seto C.
      • Hawrot E.
      ,
      • Ackermann E.J.
      • Ang E.T.-H.
      • Kanter J.R.
      • Tsigelny I.
      • Taylor P.
      ). A double-mutant cycle analysis with NmmI toxin revealed pairwise contacts between Tyr190 and R33E and R36E, with a greater coupling energy to R36E (
      • Malany S.
      • Osaka H.
      • Sine S.M.
      • Taylor P.
      ,
      • Ackermann E.J.
      • Ang E.T.-H.
      • Kanter J.R.
      • Tsigelny I.
      • Taylor P.
      ). In the present study, we document an intermolecular NOE constraint between Lys38(position corresponds to Arg36 in NmmI toxin) and Thr191, one residue removed from Tyr190 in the Bgtx·α18-mer complex.
      Cation-π interactions are interactions between a cationic group, such as the side chain of Arg and Lys, with the electronegativity of an aromatic π cloud (
      • Gallivan J.P.
      • Dougherty D.A.
      ). Cation-π interaction pairs within the Bgtx·α18-mer complex were identified using CaPTURE (
      • Gallivan J.P.
      • Dougherty D.A.
      ). Of the 120 acceptable structures, 24 showed evidence of cation-π interactions including two of the 20 best structures (Table III). One of these latter two structures was chosen to illustrate one such candidate cation-π interaction (Fig. 9). In this example, the NZ nitrogen of Bgtx Lys38 is oriented within 6 Å of the aromatic ring of Tyr190. We speculate that cation-π interactions may be involved in Bgtx binding to the nAChR just as cation-π interactions involving αTrp149 may be important in the binding of acetylcholine to the nAChR (
      • Zhong W.
      • Gallivan J.P.
      • Zhang Y.
      • Li L.
      • Lester H.A.
      • Dougherty D.A.
      ). Additional high resolution structures with better resolution of the side-chain positions would be needed to test this proposal further.
      In the Bgtx·α18-mer complex, Cys192 and Cys193 of the peptide are located in between loop I and loop II and adjacent to the C-terminal tail of Bgtx. This position correlates well with recent biochemical cross-linking data concerning the spatial orientation of the reduced disulfide bond between Cys192 and Cys193 at the αγ interface of the Torpedo nAChR (
      • Michalet S.
      • Teixeira F.
      • Gilquin B.
      • Mourier G.
      • Servent D.
      • Drevet P.
      • Binder P.
      • Tzartos S.
      • Ménez A.
      • Kessler P.
      ). Several Cys-substituted mutants ofNaja nigricollis α-neurotoxin, a short-chain neurotoxin, were cross-linked to the Torpedo α subunit with various efficiencies depending on the spacer length of the dimaleimide derivative used and the site of toxin mutagenesis. It was concluded that Cys192 and Cys193 were located under the tip of the first loop, ∼11.5 Å from the α-carbon at toxin position 10, and close to the second loop, ∼ 15.5 Å from the α-carbon at toxin position 33 (51). This orientation is entirely consistent with the structure shown in Fig. 6 B.
      In summarizing the contact information, we find that Bgtx interacts with the α18-mer primarily through three contact regions. Arg36-Val40 in loop II appears to be a core binding site that is common to both the Bgtx·α18-mer complex and the earlier Bgtx·α12-mer complex. We also find that the tip of loop I is involved in peptide binding. Lys70 and Arg72 of the C-terminal tail provide the third binding region that serves to function as a physical continuum between the two other binding sites. As described above, there is a remarkable correlation between the conclusions drawn from various mutagenesis and biochemical cross-linking studies and the structures presented here for the Bgtx·α18-mer complex. Although it is clear that additional receptor contacts are required to achieve the Bgtx binding affinity observed with the native receptor, the evidence to date nevertheless suggests that the α18-mer complex with Bgtx may serve as a useful and accessible model for studying the structural and energetic basis of the high affinity toxin-receptor interaction.

      Acknowledgments

      We thank Dr. Dale F. Mierke for critical reading of and comments on the manuscript. We acknowledge the assistance of Andrea Piserchio with the NMR experiments. We also thank Dr. Vladimir J. Basus for helpful discussions.

      REFERENCES

        • Karlin A.
        • Akabas M.H.
        Neuron. 1995; 15: 1231-1244
        • Antil S.
        • Servent D.
        • Ménez A.
        J. Biol. Chem. 1999; 274: 34851-34858
        • Malany S.
        • Osaka H.
        • Sine S.M.
        • Taylor P.
        Biochemistry. 2000; 39: 15388-15398
        • Rosenthal J.A.
        • Levandoski M.M.
        • Chang B.
        • Potts J.F.
        • Shi Q.-L.
        • Hawrot E.
        Biochemistry. 1999; 38: 7847-7855
        • Rosenthal J.A.
        • Hsu S.H.
        • Schneider D.
        • Gentile L.N.
        • Messier N.J.
        • Vaslet C.A.
        • Hawrot E.
        J. Biol. Chem. 1994; 269: 11178-11185
        • Love R.A.
        • Stroud R.M.
        Protein Eng. 1986; 1: 37-46
        • Basus V.J.
        • Billeter M.
        • Love R.A.
        • Stroud R.M.
        • Kuntz I.D.
        Biochemistry. 1988; 27: 2763-2771
        • Labhardt A.M.
        • Hunziker-Kwik E.H.
        • Wüthrich K.
        Eur. J. Biochem. 1988; 177: 295-305
        • Le-Goas R.
        • LaPlante S.R.
        • Mikou A.
        • Delsuc M.A.
        • Guittet E.
        • Robin M.
        • Charpentier I.
        • Lallemand J.Y.
        Biochemistry. 1992; 31: 4867-4875
        • Zinn-Justin S.
        • Roumestand C.
        • Gilquin B.
        • Bontems F.
        • Ménez A.
        • Toma F.
        Biochemistry. 1992; 31: 11335-11347
        • Connolly P.J.
        • Stern A.S.
        • Hoch J.C.
        Biochemistry. 1996; 35: 418-426
        • Peng S.-S.
        • Kumar T.K.S.
        • Jayaraman G.
        • Chang C.-C.
        • Yu C.
        J. Biol. Chem. 1997; 272: 7817-7823
        • Wilson P.T.
        • Lentz T.L.
        • Hawrot E.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8790-8794
        • Grutter T.
        • Ehret-Sabatier L.
        • Kotzyba-Hibert F.
        • Goeldner M.
        Biochemistry. 2000; 39: 3034-3043
        • Kachalsky S.G.
        • Jensen B.S.
        • Barchan D.
        • Fuchs S.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10801-10805
        • Wilson P.T.
        • Hawrot E.
        • Lentz T.L.
        Mol. Pharmacol. 1988; 34: 643-650
        • Pearce S.F.A.
        • Preston-Hurlburt P.
        • Hawrot E.
        Proc. R. Soc. Lond. Biol. Sci. 1990; 241: 207-213
        • Spura A.
        • Riel R.U.
        • Freedman N.D.
        • Agrawal S.
        • Seto C.
        • Hawrot E.
        J. Biol. Chem. 2000; 275: 22452-22460
        • Ackermann E.J.
        • Ang E.T.-H.
        • Kanter J.R.
        • Tsigelny I.
        • Taylor P.
        J. Biol. Chem. 1998; 273: 10958-10964
        • Tomaselli G.F.
        • McLaughlin J.T.
        • Jurman M.E.
        • Hawrot E.
        • Yellen G.
        Biophys. J. 1991; 60: 721-727
        • Galzi J.L.
        • Bertrand D.
        • Devillers-Thiery A.
        • Revah F.
        • Bertrand S.
        • Changeux J.P.
        FEBS Lett. 1991; 294: 198-202
        • Basus V.J.
        • Song G.
        • Hawrot E.
        Biochemistry. 1993; 32: 12290-12298
        • Kuliopulos A.
        • Walsh C.T.
        J. Am. Chem. Soc. 1994; 116: 4599-4607
        • Piotto M.
        • Saudek V.
        • Sklenar V.
        J. Biomol. NMR. 1992; 2: 661-666
        • Sklenar V.
        • Piotto M.
        • Leppik R.
        • Saudek V.
        J. Magn. Reson. Ser. A. 1993; 102: 241-245
        • Bodenhausen G.
        • Ruben D.J.
        Chem. Phys. Lett. 1980; 69: 185-189
        • Bax A.
        • Davis D.G.
        J. Magn. Reson. 1985; 65: 355-360
        • Davis A.L.
        • Keeler J.
        • Laue E.D.
        • Moskau D.
        J. Magn. Reson. 1992; 98: 207-216
        • Palmer III A.G.
        • Cavanagh J.
        • Wright P.E.
        • Rance M.
        J. Magn. Reson. 1991; 93: 151-170
        • Kay L.E.
        • Keifer P.
        • Saarinen T.
        J. Am. Chem. Soc. 1992; 114: 10663-10665
        • Schleucher J.
        • Sattler M.
        • Griesinger C.
        Angew. Chem. Int. Ed. Engl. 1993; 32: 1489-1491
        • Vuister G.W.
        • Bax A.
        J. Am. Chem. Soc. 1993; 115: 7772-7777
        • Vuister G.W.
        • Bax A.
        J. Biomol. NMR. 1994; 4: 193-200
        • Delaglio F.
        • Grzesiek S.
        • Vuister G.W.
        • Zhu G.
        • Pfeifer J.
        • Bax A.
        J. Biomol. NMR. 1995; 6: 277-293
        • Goddard T.D.
        • Kneller D.G.
        SPARKY 3.95. University of California, San Francisco, CA2000
        • Gentile L.N.
        • Basus V.J.
        • Shi Q.-L.
        • Hawrot E.
        Ann. N. Y. Acad. Sci. 1995; 757: 222-237
        • Bax A.
        • Vuister G.W.
        • Grzesiek S.
        • Delaglio F.
        • Wang A.C.
        • Tschudin R.
        • Zhu G.
        Methods Enzymol. 1994; 239: 79-105
        • Thompson G.S.
        • Leung Y.-C.
        • Ferguson S.J.
        • Radford S.E.
        • Redfield C.
        Protein Sci. 2000; 9: 846-858
        • Brunger 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: 905-921
        • Rölz C.
        • Mierke D.F.
        Biophys. Chem. 2001; 89: 119-128
        • Sayle R.A.
        • Milner-White E.J.
        Trends Biochem. Sci. 1995; 20: 374-376
        • Koradi R.
        • Billeter M.
        • Wüthrich K.
        J. Mol. Graph. 1996; 14: 51-55
        • Sobolev V.
        • Sorokine A.
        • Prilusky J.
        • Abola E.E.
        • Edelman M.
        Bioinformatics. 1999; 15: 327-332
        • Gallivan J.P.
        • Dougherty D.A.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9459-9464
        • Feng S.
        • Chen J.K., Yu, H.
        • Simon J.A.
        • Schreiber S.L.
        Science. 1994; 266: 1241-1247
        • Goldenberg D.P.
        • Koehn R.E.
        • Gilbert D.E.
        • Wagner G.
        Protein Sci. 2001; 10: 538-550
        • Scherf T.
        • Balass M.
        • Fuchs S.
        • Katchalski-Katzir E.
        • Anglister J.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6059-6064
        • Wu S.-H.
        • Chen C.-J.
        • Tseng M.-J.
        • Wang K.-T.
        Arch. Biochem. Biophys. 1983; 227: 111-117
        • Levandoski M.M.
        • Lin Y.
        • Moise L.
        • McLaughlin J.T.
        • Cooper E.
        • Hawrot E.
        J. Biol. Chem. 1999; 274: 26113-26119
        • Zhong W.
        • Gallivan J.P.
        • Zhang Y.
        • Li L.
        • Lester H.A.
        • Dougherty D.A.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12088-12093
        • Michalet S.
        • Teixeira F.
        • Gilquin B.
        • Mourier G.
        • Servent D.
        • Drevet P.
        • Binder P.
        • Tzartos S.
        • Ménez A.
        • Kessler P.
        J. Biol. Chem. 2000; 275: 25608-25615