We report here the solution structure of omega-conotoxin GVIA, a peptide antagonist of the N-type neuronal voltage-sensitive calcium channel. The structure was determined using two-dimensional NMR in combination with distance geometry and restrained molecular dynamics. The full relaxation matrix analysis program MARDIGRAS was used to generate maximum and minimum distance restraints from the crosspeak intensities in NOESY spectra. The 187 restraints obtained were used in conjunction with 23 angle restraints from vicinal coupling constants as input for the structure calculations. The backbones of the best 21 structures match with an average pairwise RMSD of 0.58 A. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 18-21, and 24-27, making this the smallest published peptide structure to contain a triple-stranded beta-sheet. Conotoxins have been shown to be effective neuroprotective agents in animal models of brain ischemia. Our results should aid in the design of novel nonpeptide compounds with potential therapeutic utility.

Solution structures for many proteins have been determined to date utilizing interproton distance constraints estimated from two-dimensional nuclear Overhauser effect (2D NOE) spectra. Although the simple isolated spin pair approximation (ISPA) generally used can result in systematic errors in distances, the large number of constraints enables protein structure to be defmed with reasonably high resolution. Effects of these systematic errors on the resulting protein structure are examined. Iterative relaxation matrix calculations, which account for dipolar interactions between all protons in a molecule, can accurately determine internuclear distances with little or no a priori knowledge of the molecular structure. The value of this additional complexity is also addressed. To assess these distance determination methods, hypothetical "experimental" data, including random noise and peak overlap, are calculated for an arbitrary "true" protein structure. Three methods of obtaining distance constraints from 2D NOE peak intensities are examined: one entails a conservative use of ISPA, one assumes the ISPA to be fairly accurate, and one utilizes an iterative relaxation matrix method called MARDIGRAS (matrix analysis of relaxation for discerning the geometry of an aqueous structure), developed in this laboratory. A distance geometry algorithm was used to generate a family of structures for each distance set. The quality of the average structure from each family was good. The rootmean-square deviation of that average structure from the true structure was improved about 2-5% using the more restrictive rather than the more conservative ISPA approach. Use of MARDIGRAS in a conservative fashion-i.e., with a poor initial model-resulted in improvement in the root-mean-square deviation by 8-15%. With a better initial model, MARDIGRAS obtained even more accurate distances. MARDIGRAS also permits analysis of 2D NOE data at longer mixing times, yielding additional distances. Use of more restrictive ISPA distances did, however, result in a few systematically incorrect structural features in local regions of the protein, producing distortions of 2-3 A. Comparison between experimental data and spectra calculated for the structures correlates with root-mean-square deviation, offering a method of structure evaluation. An R factor for evaluating fit between experimental and calculated 2D NOE intensities is proposed.Interproton distances obtained from homonuclear proton two-dimensional nuclear Overhauser effect (2D NOE) experiments are used to determine three-dimensional protein structure in solution (1-4). Various protocols are used for structure determination, but the initial step often utilizes distance geometry (DG) to generate a family of structures consistent with NOE distance constraints (5, 6). Some methods entail theoretical energy calculations, energy minimization, or restrained molecular dynamics, with pseudoenergy terms maintaining NOE-derived distances (7,8). Families of structures for several proteins have been produc...

We report the two-dimensional nuclear magnetic resonance (NMR) characterization of the stoichiometric complex formed between the snake venom-derived long alpha-neurotoxin, alpha-bungarotoxin (BGTX), and a synthetic dodecapeptide (alpha 185-196) corresponding to a functionally important region on the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) obtained from Torpedo californica electric organ tissue. BGTX has been widely used as the classic nicotinic competitive antagonist for the skeletal muscle type of nAChR which is found in the avian, amphibian, and mammalian neuromuscular junction. The receptor dodecapeptide (alpha 185-196) binds BGTX with micromolar affinity and has been shown to represent the major determinant of BGTX binding to the isolated alpha-subunit. Previous studies involving covalent modification of the native nAChR from Torpedo membranes with a variety of affinity reagents indicate that several residues contained within the dodecapeptide sequence (namely, Tyr-190, Cys-192, and Cys-193) apparently contribute directly to the formation of the cholinergic ligand binding site. The NMR-derived solution structure of the BGTX/receptor peptide complex defines a relatively extended conformation for a major segment of the "bound" dodecapeptide. These structural studies also reveal a previously unpredicted receptor binding cleft within BGTX and suggest that BGTX undergoes a conformational change upon peptide binding. If, as we hypothesize, the identified intermolecular contacts in the BGTX/receptor peptide complex describe a portion of the contact zone between BGTX and native receptor, then the structural data would suggest that alpha-subunit residues 186-190 are on the extracellular surface of the receptor.

We report a new, higher resolution NMR structure of ␣-bungarotoxin that defines the structure-determining disulfide core and ␤-sheet regions. We further report the NMR structure of the stoichiometric complex formed between ␣-bungarotoxin and a recombinantly expressed 19-mer peptide ( The nicotinic acetylcholine receptor (nAChR)1 (1) is a ligandgated 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, ␥-aminobutyric acid type A, ␥-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 (␣1) 2 ␤␥␦ and (␣1) 2 ␤⑀␦ in embryonic and adult tissue, respectively (for review, see Ref.

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