Three-Dimensional Solution Structure of Conotoxin ψ-PIIIE, an Acetylcholine Gated Ion Channel Antagonist †,‡ Scott S. Mitchell, § Ki Joon Shon, | Mark P. Foster, § Darrell R. Davis, § Baldomero M. Olivera, ⊥ and Chris M. Ireland* ,§ Department of Medicinal Chemistry, UniVersity of Utah, Salt Lake City, Utah 84112, Department of Biology, UniVersity of Utah, Salt Lake City, Utah 84112, and Department of Physiology and Biophysics, Case Western ReserVe UniVersity, 10900 Euclid AVenue, CleVeland, Ohio 44106 ReceiVed September 3, 1997; ReVised Manuscript ReceiVed October 31, 1997 ABSTRACT: The three-dimensional structure of conotoxin ψ-PIIIE, a 24-amino acid peptide from Conus purpurascens, has been solved using two-dimensional (2D) 1 H NMR spectroscopy. Conotoxin ψ-PIIIE contains the same disulfide bonding pattern as the µ-conotoxins, which target skeletal muscle sodium channels, but has been shown to antagonize the acetylcholine gated cation channel through a noncompetitive mechanism. Structural information was obtained by the analysis of a series of 2D NOESY spectra as well as measurement of coupling constants from 1D 1 H and PE-COSY NMR experiments. Molecular modeling calculations included the use of the distance geometry (DG) algorithm, simulated annealing techniques, and the restrained molecular dynamics method. The resulting structures are considerably similar to the previously published structures for the µ-conotoxins GIIIA and GIIIB, despite the lack of sequence conservation between conotoxin ψ-PIIIE and the µ-conotoxins. The structure consists of a series of tight turns, each turn occurring in the position analogous to those of turns described in µ-GIIIA and µ-GIIIB. This suggests the disulfide bonding pattern is able to largely direct the structure of the peptides, creating a stable structural motif which allows extensive sequence substitution of non-cystine residues. Conotoxin ψ-PIIIE is a recently described peptide toxin from the venom of the venomous snail Conus purpurascens that has been shown to act as a noncompetitive inhibitor of the acetylcholine receptor (1). ψ-PIIIE contains the same disulfide bonding pattern as the µ-conotoxins, though it shares no other sequence homology with this pharmacologi- cal class of peptides (Figure 1). Three-dimensional structures and analysis of structure-activity relationships have recently been published for the µ-conotoxins GIIIA (2-4) and GIIIB (5) describing a compact structure built around a cage of disulfide-bonded sulfur atoms. Replacement of individual non-cystine residues with alanine demonstrated voltage gated sodium channel binding activity for µ-GIIIA was particularly sensitive to mutations at residue R13 (6). This residue is not conserved between the sequences of µ-GIIIA and ψ-PIIIE, and the three-dimensional structure of ψ-PIIIE does not contain an equivalent functional group replacing the critical guanidinium group of R13 in µ-GIIIA. This result partially explains the altered binding specificity between the two peptides. The three-dimensional structure of conotoxin ψ-PIIIE is of particular interest, as differences in the structures will be useful in describing the structural requirements of the binding sites on the voltage gated sodium and the acetylcholine gated ion channels. EXPERIMENTAL PROCEDURES Sample. The peptide was synthesized using FMOC 1 solid phase peptide synthesis techniques. Cysteine residues were oxidized with glutathione as previously described (7). The resulting isomers were separated using reversed phase HPLC and tested for both biological activity and HPLC comigration with the native compound. The biological activity of the resulting material was found to be equivalent with that of the natural product. NMR Spectroscopy. All spectra were recorded on a Varian Unity 500 MHz spectrometer equipped with a triple-channel waveform generator. The sample was prepared by dissolving 6 mg of the peptide in 475 µL of 90% H 2 O/10% D 2 O and † Supported by NIH Grants P01 GM48677 and GM54710. ‡ Coordinates for the final structures have been deposited at the Brookhaven Protein Data Bank, Upton, NY 11973, under accession code 1as5. § Department of Medicinal Chemistry, University of Utah. | Case Western Reserve University. ⊥ Department of Biology, University of Utah. 1 Abbreviations: 2D 1 H NMR, two-dimensional proton nuclear magnetic resonance spectroscopy; O, trans-4-hydroxyproline; NOESY, nuclear Overhauser effect spectroscopy; PE-COSY, primitive exclusive correlation spectroscopy; DQF-COSY, double-quantum filtered cor- relation spectroscopy; TOCSY, total correlation spectroscopy; DG, distance geometry; RMD, restrained molecular dynamics; RMA, relaxation matrix approach; IRMA, iterative relaxation matrix approach; T 1, longitudinal relaxation time; RMSD, root-mean-squared deviation; FMOC, fluorenylmethoxycarbonyl. FIGURE 1: Aligned sequences of peptides µ-GIIIA, µ-GIIIB, and ψ-PIIIE showing disulfide bond connectivities. The C terminus of each peptide is amidated. 1215 Biochemistry 1998, 37, 1215-1220 S0006-2960(97)02186-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/03/1998