Solution structure of delta-Am2766: a highly hydrophobic delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.     

Three-dimensional solution structure of omega-conotoxin TxVII, an L-type calcium channel blocker

We determined the three-dimensional structure of omega-conotoxin TxVII, a 26-residue peptide that is an L-type calcium channel blocker, by (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 411 distance constraints obtained from nuclear Overhauser effect connectivities, 20 torsion angle constraints, and 21 constraints associated with hydrogen bonds and disulfide bonds. The root-mean-square deviations about the averaged coordinates of the backbone atoms (N, C(alpha), C, and O) and all heavy atoms were 0.50 +/- 0.09 A and 0.99 +/- 0.13 A, respectively. The structure of omega-conotoxin TxVII is composed of a triple-stranded antiparallel beta-sheet and four turns. The three disulfide bonds in omega-conotoxin TxVII form the classical cystine knot motif of toxic or inhibitory polypeptides. The overall folding of omega-conotoxin TxVII is similar to those of the N-type calcium channel blockers, omega-conotoxin GVIA and MVIIA, despite the low amino acid sequence homology among them. omega-Conotoxin TxVII exposes many hydrophobic residues to a certain surface area. In contrast, omega-conotoxin GVIA and MVIIA expose basic residues in the same way as omega-conotoxin TxVII. The channel binding site of omega-conotoxin TxVII is different from those of omega-conotoxin GVIA and MVIIA, although the overall folding of these three peptides is similar. The gathered hydrophobic residues of omega-conotoxin TxVII probably interact with the hydrophobic cluster of the alpha(1) subunit of the L-type calcium channel, which consists of 13 residues located in segments 5 and 6 in domain III and in segment 6 in domain IV.     

Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

Solution structure of toxin 2 from centruroides noxius Hoffmann, a beta-scorpion neurotoxin acting on sodium channels

We have determined the solution structure of Cn2, a beta-toxin extracted from the venom of the New World scorpion Centruroides noxius Hoffmann. Cn2 belongs to the family of scorpion toxins that affect the sodium channel activity, and is very toxic to mammals (LD50=0.4 microg/20 g mouse mass). The three-dimensional structure was determined using 1H-1H two-dimensional NMR spectroscopy, torsion angle dynamics, and restrained energy minimization. The final set of 15 structures was calculated from 876 experimental distance constraints and 58 angle constraints. The structures have a global r. m.s.d. of 1.38 A for backbone atoms and 2.21 A for all heavy atoms. The overall fold is similar to that found in the other scorpion toxins acting on sodium channels. It is made of a triple-stranded antiparallel beta-sheet and an alpha-helix, and is stabilized by four disulfide bridges. A cis-proline residue at position 59 induces a kink of the polypeptide chain in the C-terminal region. The hydrophobic core of the protein is made up of residues L5, V6, L51, A55, and by the eight cysteine residues. A hydrophobic patch is defined by the aromatic residues Y4, Y40, Y42, W47 and by V57 on the side of the beta-sheet facing the solvent. A positively charged patch is formed by K8 and K63 on one edge of the molecule in the C-terminal region. Another positively charged spot is represented by the highly exposed K35. The structure of Cn2 is compared with those of other scorpion toxins acting on sodium channels, in particular Aah II and CsE-v3. This is the first structural report of an anti-mammal beta-scorpion toxin and it provides the necessary information for the design of recombinant mutants that can be used to probe structure-function relationships in scorpion toxins affecting sodium channel activity.     

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 2. Three-dimensional solution structure.

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D 1H NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and muO-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18+/-0.05 A for the backbone atoms and 1.39+/-0.33 A for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.     

Delta-conotoxin structure/function through a cladistic analysis.

Delta-conotoxins are Conus peptides that inhibit inactivation of voltage-gated sodium channels. The suggestion that delta-conotoxins might be an essential component of the venoms of fish-hunting cone snails which rapidly immobilize their prey [Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151] has not been tested. On the basis of cDNA cloning, all of the fish-hunting Conus analyzed yielded at least one delta-conotoxin sequence. In addition, one delta-conotoxin isolated from the venom of Conus striatus had an amino acid sequence identical to that predicted from cDNA cloning. This new peptide exhibited properties of delta-conotoxins: it targeted sodium channels and potentiated action potentials by slowing channel inactivation. Homologous sequences of delta-conotoxins from two groups (clades) of related fish-hunting Conus species share consensus features but differ significantly from the two known delta-conotoxins from mollusc-hunting Conus venoms. Three large hydrophobic amino acids were conserved; analogues of the previously described delta-conotoxin PVIA with alanine substituted for the conserved amino acids F9 and I12 lost substantial biological activity. In contrast, both the T8A and K13A delta-conotoxin PVIA analogues, where substitutions were at nonconserved loci, proved to be biologically active. Taken together, our results indicate that a cladistic approach can identify amino acids critical for the activity of conotoxins and provide extensive information as to which amino acid substitutions can be made without significant functional consequences.     

The three-dimensional structure of guanine-specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry.

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Solution structure of ZipA, a crucial component of Escherichia coli cell division

ZipA, an essential component of cell division in Escherichia coli, interacts with the FtsZ protein at the midcell in one of the initial steps of septum formation. The high-resolution solution structure of the 144-residue C-terminal domain of E. coli ZipA (ZipA(185)(-)(328)) has been determined by multidimensional heteronuclear NMR. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2758 experimental NMR restraints. The atomic root means square distribution about the mean coordinate positions for residues 6-142 for the 30 structures is 0.37 +/- 0.04 A for the backbone atoms, 0. 78 +/- 0.05 A for all atoms, and 0.45 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of ZipA(185)(-)(328) is composed of three alpha-helices and a beta-sheet consisting of six antiparallel beta-strands where the alpha-helices and the beta-sheet form surfaces directly opposite each other. A C-terminal peptide from FtsZ has been shown to bind ZipA(185)(-)(328) in a hydrophobic channel formed by the beta-sheet providing insight into the ZipA-FtsZ interaction. An unexpected similarity between the ZipA(185)(-)(328) fold and the split beta-alpha-beta fold observed in many RNA binding proteins may further our understanding of the critical ZipA-FtsZ interaction.     

Synthesis, bioactivity, and cloning of the L-type calcium channel blocker omega-conotoxin TxVII.

omega-Conotoxin TxVII is the first conotoxin reported to block L-type currents. In contrast to other omega-conotoxins, its sequence is characterized by net negative charge and high hydrophobicity, although it retains the omega-conotoxin cysteine framework. In order to obtain structural information and to supply material for further characterization of its biological function, we synthesized TxVII and determined its disulfide bond pairings. Because a linear precursor with free SH groups showed a strong tendency to aggregate and to polymerize, we examined many different conditions for air oxidation and concluded that a mixture of cationic buffer and hydrophobic solvent was the most effective for the folding of TxVII. Synthetic TxVII was shown to suppress the slowly inactivating voltage-dependent calcium current in cultured Lymnaea RPeD1 neurons and furthermore to suppress synaptic transmission between these neurons and their follower cells. In contrast, TxVII did not block calcium flux through L-type channels in PC12 cells, suggesting a phyletic or subtype specificity in this channel family. Disulfide bond pairings of TxVII and its isomers were determined by enzymatic fragmentation in combination with chemical synthesis, thus revealing that TxVII has the same disulfide bond pattern as other omega-conotoxins. Furthermore, the CD spectrum of TxVII is similar to those of omega-conotoxins MVIIA and MVIIC. The precursor sequence of TxVII was determined by cDNA cloning and shown to be closest to that of delta-conotoxin TxVIA, a sodium channel inactivation inhibitor. Thus TxVII conserves the structural fold of other omega-conotoxins, and the TxVIA/TxVII branch of this family reveals the versatility of its structural scaffold, allowing evolution of structurally related peptides to target different channels.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

Solution structure of Pisum sativum defensin 1 by high resolution NMR: plant defensins, identical backbone with different mechanisms of action.

Pisum sativum defensin 1 (Psd1) is a 46 amino acid residue plant defensin isolated from seeds of pea. The three-dimensional structure in solution of Psd1 was determined by two-dimensional NMR data recorded at 600 MHz. Experimental restraints were used for structure calculation using CNS and torsion-angle molecular dynamics. The 20 lowest energy structures were selected and further subjected to minimization, giving a root-mean-square deviation of 0.78(+/- 0.22) A in the backbone and 1.91(+/-0.60) A for over all atoms of the molecule. The protein has a globular fold with a triple-stranded antiparalell beta-sheet and an alpha-helix (from residue Asn17 to Leu27). Psd1 presents the so called "cysteine stabilized alpha/beta motif" and presents identical three-dimensional topology in the backbone with other defensins and neurotoxins. Comparison of the electrostatic surface potential among proteins with high three-dimensional (selected using the softwares TOP and DALI) topology gave insights into the mode of action of Psd1. The surface topologies between proteins that present antifungal activity or sodium channel inhibiting activity are different. On the other hand the surface topology presents several common features with potassium channel inhibitors, suggesting that Psd1 presents this activity. Other common features with potassium channel inhibitors were found including the presence of a lysine residue essential for inhibitory activity. The identity of Psd1 in primary sequence is not enough to infer a mechanism of action, in contrast with the strategy proposed here.     

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds. Agelenin has characteristic residues, Phe9, Ser28 and Arg33, which are arranged similarly to the pharmacophore of the insect channel inhibitor, omega-atracotoxin-Hv1a. These observations suggest that agelenin and omega-atracotoxin-Hv1a bind to insect calcium channels in a similar manner. We also suggest that another mode of action may operate in the channel inhibition by omega-agatoxin-IVA and omega-atracotoxin-Hv2a.     

NMR structure of the Euplotes raikovi pheromone Er-23 and identification of its five disulfide bonds.

The NMR solution structure of the 51 residue pheromone Er-23 from the ciliated protozoan Euplotes raikovi (Er) was calculated with the torsion angle dynamics program DYANA from 582 nuclear Overhauser enhancement (NOE) upper limit distance constraints, 46 dihedral angle constraints and 30 disulfide bond constraints. The disulfide bridges had not been assigned by chemical methods, and initially were assigned tentatively on the basis of inspection of the positioning of the Cys sulfhydryl groups in a bundle of 20 conformers that was calculated without disulfide bond constraints. The assignment of disulfide bridges was then validated by structure calculations that assessed the compatibility of plausible alternative Cys-Cys disulfide combinations with the input of NOE upper distance constraints and dihedral angle constraints. For a group of 20 conformers used to characterize the solution structure, the average pairwise root-mean-square distances from the mean coordinates calculated for the backbone heavy atoms N, C(alpha) and C' of resideus 1-51 is 0.38 A. The molecular architecture consists of a three-dimensional arrangement of five helices comprised of residues 2-8, 14-17, 26-29, 34-36 and 38-47, with five disulfide bridges in the positions 3-24, 6-16, 13-47, 27-40, and 35-51, which has so far not been represented in the Protein Data Bank. Er-23 is unique among presently known Er-pheromones with respect to size, sequence, the number of disulfide bonds and the three-dimensional structure, thus providing a new structural basis for rationalizing the physiological functions of this protein family.     

Interactions of δ-Conotoxins with Alkaloid Neurotoxins Reveal Differences Between the Silent and Effective Binding Sites on Voltage-Sensitive Sodium Channels

Abstract: The δ-conotoxin-TxVIA from Conus textile (δTxVIA) is a mollusk-specific conotoxin that slows sodium channel inactivation exclusively in mollusk neuronal membranes but reveals high-affinity binding to both mollusk (effective binding) and rat brain (silent binding) neuronal membranes, despite not having any toxic effect in vertebrates in vivo and in vitro. Using binding studies with radioactive δTxVIA we demonstrate that a different mollusk-specific conotoxin, δ-conotoxin-GmVIA from the venom of Conus gloriamaris , possesses "silent" and effective binding properties in rat brain and mollusk sodium channels, respectively. Binding studies and electrophysiological tests with both vertebrate muscle and insect neuronal preparations have indicated that the silent binding sites of δTxVIA are highly conserved in a wide range of distinct vertebrate and insect sodium channels. Direct probing of receptor site 2 by a tritiated derivative of batrachotoxin ([³H]BTX-B) revealed that [³H]BTX-B binding in mollusk sodium channels is of high affinity with no addition of enhancing ligands, unlike [³H]BTX-B binding in rat brain. In contrast to the negative allosteric modulation of δTxVIA binding by veratridine, δTxVIA is not able to affect the binding of [³H]BTX-B in mollusk neuronal membranes but reduces [³H]BTX-B binding in rat brain in the presence of α-scorpion toxins. The latter finding indicates the existence of a pharmacological distinction between the silent and effective binding sites of δTxVIA and points out possible functionally important structural differences between molluscan and rat brain sodium channels.     

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.     

Tertiary structure of conotoxin GIIIA in aqueous solution

The three-dimensional structure of conotoxin GIIIA, an important constituent of the venom from the marine hunting snail Conus geographus L., was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based methods. On the basis of 162 assigned nuclear Overhauser effect (NOE) connectivities obtained at the medium field strength frequency of 400 MHz, 74 final distance constraints of sequential and tertiary ones were derived and used together with 18 torsion angle (phi, chi 1) constraints and 9 distance constraints derived from disulfide bridges. A total of 32 converged structures were obtained from 200 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding the terminal residues 1 and 22) is 0.8 A for backbone atoms (N, C alpha, C). Conotoxin GIIIA is characterized by a particular folding of the 22 amino acid peptidic chain, which is stabilized by three disulfide bridges arranged in cage at the center of a discoidal structure of approximately 20-A diameter. The seven cationic side chains of lysine and arginine residues project radially into the solvent and form potential sites of interaction with the skeletal muscle sodium channel for which the toxin is a strong inhibitor. The present results provide a molecular basis to elucidate the remarkable physiological properties of this neurotoxin.     

Solution structure of a K+-channel blocker from the scorpion Tityus cambridgei

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.     

Structure-activity relationships of mu-conotoxin GIIIA: structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations.

A synthetic replacement study of the amino acid residues of mu-conotoxin GIIIA, a peptide blocker for muscle sodium channels, has recently shown that the conformation formed by three disulfide bridges and the molecular basicity, especially the one around the Arg13 residue, are important for blocking activity. In the present study, we determined the three-dimensional structure of an inactive analog, [Ala13]mu-conotoxin GIIIA, and refined that of the native toxin by NMR spectroscopy combined with simulated annealing calculations. The atomic root-mean-square difference of the mutant from the native conotoxin was 0.62 A for the backbone atoms (N, C alpha, C') of all residues except for the two terminal residues. The observation that the replacement of Arg13 by Ala13 does not significantly change the molecular conformation suggests that the loss of activity is not due to the conformational change but to the direct interaction of the essential Arg13 residue with the sodium channel molecules. In the determined structure, important residues for the activity, Arg13, Lys16, Hyp(hydroxyproline)17, and Arg19, are clustered on one side of the molecule, an observation which suggests that this face of the molecule associates with the receptor site of sodium channels. The hydroxyl group of Hyp17 is suggested to interact with the channel site with which the essential hydroxyl groups of tetrodotoxin and saxitoxin interact.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues 20-83) of barley serine proteinase inhibitor 2 (BSPI-2) is determined on the basis of 403 interproton distance, 34 phi backbone torsion angle and 26 hydrogen bonding restraints derived from n.m.r. measurements. A total of 11 converged structures were computed using a metric matrix distance geometry algorithm and refined by restrained molecular dynamics. The average rms difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.2 A for the backbone atoms and 2.1 +/- 0.1 A for all atoms. The overall structure, which is almost identical to that found by X-ray crystallography, is disc shaped and consists of a central four component mixed parallel and antiparallel beta-sheet flanked by a 13 residue alpha-helix on one side and the reactive site loop on the other.