Brownian dynamics simulations of interaction between scorpion toxin Lq2 and potassium ion channel

The association of the scorpion toxin Lq2 and a potassium ion (K(+)) channel has been studied using the Brownian dynamics (BD) simulation method. All of the 22 available structures of Lq2 in the Brookhaven Protein Data Bank (PDB) determined by NMR were considered during the simulation, which indicated that the conformation of Lq2 affects the binding between the two proteins significantly. Among the 22 structures of Lq2, only 4 structures dock in the binding site of the K(+) channel with a high probability and favorable electrostatic interactions. From the 4 candidates of the Lq2-K(+) channel binding models, we identified a good three-dimensional model of Lq2-K(+) channel complex through triplet contact analysis, electrostatic interaction energy estimation by BD simulation and structural refinement by molecular mechanics. Lq2 locates around the extracellular mouth of the K(+) channel and contacts the K(+) channel using its beta-sheet rather than its alpha-helix. Lys27, a conserved amino acid in the scorpion toxins, plugs the pore of the K(+) channel and forms three hydrogen bonds with the conserved residues Tyr78(A-C) and two hydrophobic contacts with Gly79 of the K(+) channel. In addition, eight hydrogen-bonds are formed between residues Arg25, Cys28, Lys31, Arg34 and Tyr36 of Lq2 and residues Pro55, Tyr78, Gly79, Asp80, and Tyr82 of K(+) channel. Many of them are formed by side chains of residues of Lq2 and backbone atoms of the K(+) channel. Thirteen hydrophobic contacts exist between residues Met29, Asn30, Lys31 and Tyr36 of Lq2 and residues Pro55, Ala58, Gly79, Asp80 and Tyr82 of the K(+) channel. These favorable interactions stabilize the association between the two proteins. These observations are in good agreement with the experimental results and can explain the binding phenomena between scorpion toxins and K(+) channels at the level of molecular structure. The consistency between the BD simulation and the experimental data indicates that our three-dimensional model of Lq2-K(+) channel complex is reasonable and can be used in further biological studies such as rational design of blocking agents of K(+) channels and mutagenesis in both toxins and K(+) channels.     

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases.

Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.     

Determination of the three-dimensional structure of iberiotoxin in solution by 1H nuclear magnetic resonance spectroscopy.

The solution structure of chemically synthesized iberiotoxin, a scorpion toxin that blocks Ca(2+)-activated K+ channels, has been determined using 2D 1H NMR spectroscopy. Analysis of the NOEs, coupling constants, and HN-DN exchange rates indicates the structure consists of an antiparallel beta-sheet from residues 25 to 36, with a type 1 turn at residues 30-31, and a helix from residues 13 to 21. The carboxyl-terminal residues form a short, and distorted, third strand of the sheet. The NMR data are consistent with disulfide bonds from residues 7 to 28, 13 to 33, and 17 to 35. The disulfide bridging presents the same profile as in other scorpion toxins, where a Cys-X-Cys sequence in a strand of sheet forms two disulfide bonds to a Cys-X-X-X-Cys sequence in a helix. Three-dimensional structures were generated using the torsion angle space program PEGASUS. The best ten structures had an average rmsd over all pairwise comparisons of 1.49 A. The average rmsd to a calculated average structure is 1.0 A. The resulting structures appear very similar to those of charybdotoxin, a related scorpion toxin.     

Structural homology in the amino-terminal domains of two aminoacyl-tRNA synthetases

The three-dimensional structures of two animoacyl-tRNA synthetases, the methionyl-tRNA synthetase from Escherichia coli (MetRS) and the tyrosyl-tRNA synthetase from Bacillus stearothermophilus (TyrRS), show a remarkable similarity over a span of about 140 amino acids. The region of homologous folding corresponds to a five-stranded parallel beta-sheet, including a mononucleotide-binding fold. One cysteine and two histidine residues that were found to be invariant in the amino acid sequences occupy similar places in the nucleotide-binding fold. In TyrRS, these residues are close to the adenylate binding site, and in MetRS to the Mg2+-ATP binding site.     

Exploring structural features of the interaction between the scorpion toxinCnErg1 and ERG K+ channels

The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruro?des noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.     

Light at the end of the Ca(2+)-release channel tunnel: structures and mechanisms involved in ion translocation in ryanodine receptor channels

RyR and InsP3R are Ca(2+)-release channels. When induced to open by the appropriate stimulus, these channels allow Ca2+ to leave intracellular storage organelles at an astonishing rate. Investigations of the ion-handling properties of isolated RyR channels have demonstrated that, at least in comparison to voltage-gated channels of surface membranes, these channels display limited powers of discrimination between physiologically relevant cations and this relative lack of selectivity is likely to contribute to the ability of Ca(2+)-release channels to maintain high rates of cation translocation without compromising function. A range of ion-handling properties in RyR are consistent with the proposal that this channel functions as a single-ion channel and theoretical considerations indicate that the high rates of ion translocation monitored for RyR would require the pore of such a structure to be short and possess a large capture radius. Measurements of the dimensions of regions of RyR involved in ion conduction and discrimination indicate that this is likely to be the case. In each monomer of RyR/InsP3R, residues making up the last two trans-membrane spanning domains and a luminal loop linking these two helices contribute to the formation of the channel pore. The luminal loops of both RyR and InsP3R contain amino acid sequences similar to those known to form the selectivity filter of K+ channels. In addition the luminal loops of both Ca(2+)-release channels contain sequences that are likely to form helices that may be analogous to the pore helix visualised in KcsA. The correlation in structural elements of the luminal loops of RyR/InsP3R and KcsA has prompted us to speculate on the tertiary arrangement for this region of the Ca(2+)-release channels using the established structure of KcsA as a framework.     

X-ray analyses of aspartic proteinases. The three-dimensional structure at 2.1 A resolution of endothiapepsin

The molecular structure of endothiapepsin (EC 3.4.23.6), the aspartic proteinase from Endothia parasitica, has been refined to a crystallographic R-factor of 0.178 at 2.1 A resolution. The positions of 2389 protein non-hydrogen atoms have been determined and the present model contains 333 solvent molecules. The structure is bilobal, consisting of two predominantly beta-sheet domains that are related by an approximate 2-fold axis. Of approximately 170 residues, 65 are topologically equivalent when one lobe is superimposed on the other. Twenty beta-strands are arranged as five beta-sheets and are connected by regions involving 29 turns and four helices. A central sheet involves three antiparallel strands from each lobe organized around the dyad axis. Each lobe contains a further local dyad that passes through two sheets arranged as a sandwich and relates two equivalent motifs of four antiparallel strands (a, b, c, d) followed by a helix or an irregular helical region. Sheets 1N and 1C, each contain two interpenetrating psi structures contributed by strands c,d,d' and c',d',d, which are related by the intralobe dyad. A further sheet, 2N or 2C, is formed from two extended beta-hairpins from strands b,c and b',c' that fold above the sheets 1N and 1C, respectively, and are hydrogen-bonded around the local intralobe dyad. Asp32 and Asp215 are related by the interlobe dyad and form an intricate hydrogen-bonded network with the neighbouring residues and comprise the most symmetrical part of the structure. The side-chains of the active site aspartate residues are held coplanar and the nearby main chain makes a "fireman's grip" hydrogen-bonding network. Residues 74 to 83 from strands a'N and b'N in the N-terminal lobe form a beta-hairpin loop with high thermal parameters. This "flap" projects over the active site cleft and shields the active site from the solvent region. Shells of water molecules are found on the surface of the protein molecule and large solvent channels are observed within the crystal. There are only three regions of intermolecular contacts and the crystal packing is stabilized by many solvent molecules forming a network of hydrogen bonds. The three-dimensional structure of endothiapepsin is found to be similar to two other fungal aspartic proteinases, penicillopepsin and rhizopuspepsin. Even though sequence identities of endothiapepsin with rhizopuspepsin and penicillopepsin are only 41% and 51%, respectively, a superposition of the three-dimensional structures of these three enzymes shows that 237 residues (72%) are within a root-mean-square distance of 1.0 A.     

Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains

The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Our previous work showed that substitutions of equivalent residues L293 and A428 within the pore-loops had qualitatively similar effects on both the [K+]o-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity. The mutation N410D slowed current activation in a [K+]o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+. The N275D mutant, by contrast, had little effect on the [K+]o-sensitivity for activation and it greatly altered the. [K+]o-dependence of current deactivation. Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating. The double mutant N410D-N275D showed characteristics of N410D in the [K+]o-sensitivity of current activation and of N275D in the [K+]o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites. The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions.     

Solution structure of CnErg1 (Ergtoxin), a HERG specific scorpion toxin

The three-dimensional structure of chemically synthesized CnErg1 (Ergtoxin), which specifically blocks HERG (human ether-a-go-go-related gene) K+ channels, was determined by nuclear magnetic resonance spectroscopy. CnErg1 consists of a triple-stranded beta-sheet and an alpha-helix, as is typical of K+ channel scorpion toxins. The peptide structure differs from the canonical structures in that the first beta-strand is shorter and is nearer to the second beta-strand rather than to the third beta-strand on the C-terminus. There is also a large hydrophobic patch on the surface of the toxin, surrounding a central lysine residue, Lys13. We postulate that this hydrophobic patch is likely to form part of the binding surface of the toxin.     

Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry.

The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel beta-sheet and a single helix covering one face of the beta-sheet. The cysteine side chains connecting the beta-sheet and the helix form the core of the molecule. One edge of the beta-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, Arg24, Lys27, and Arg31 of the beta-sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions.     

Two rings of negative charges in the cytosolic vestibule of type-1 ryanodine receptor modulate ion fluxes

The tetrameric ryanodine receptor calcium release channels (RyRs) are cation-selective channels that have pore architecture similar to that of K+ channels. We recently identified, in close proximity to the selectivity filter motif GGGIG, a conserved lumenal DE motif that has a critical role in RyR ion permeation and selectivity. Here, we substituted three aspartate residues (D4938, D4945, D4953) with asparagine and four glutamate residues (E4942, E4948, E4952, E4955) with glutamine hypothesized to line the cytosolic vestibule of the skeletal muscle RyR (RyR1). Mutant single channel properties were determined using the planar lipid bilayer method. Two mutants (D4938N, D4945N) showed a reduced K+ ion conductance, with D4938N also exhibiting a reduced selectivity for Ca2+ compared to K+. The cytosolic location of D4938 and D4945 was confirmed using the polycation neomycin. Both D4938N and D4945N exhibited an attenuated block by neomycin to a greater extent from the cytosolic than lumenal side. By comparison, charge neutralization of lumenal loop residues (D4899Q, E4900N) eliminated the block from the lumenal but not the cytosolic side. The results suggest that, in addition to negatively charged residues on the lumenal side, rings of four negative charges formed by D4938 and D4945 in the cytosolic vestibule determine RyR ion fluxes.     

Canonical structures for the hypervariable regions of immunoglobulins

We have analysed the atomic structures of Fab and VL fragments of immunoglobulins to determine the relationship between their amino acid sequences and the three-dimensional structures of their antigen binding sites. We identify the relatively few residues that, through their packing, hydrogen bonding or the ability to assume unusual phi, psi or omega conformations, are primarily responsible for the main-chain conformations of the hypervariable regions. These residues are found to occur at sites within the hypervariable regions and in the conserved beta-sheet framework. Examination of the sequences of immunoglobulins of unknown structure shows that many have hypervariable regions that are similar in size to one of the known structures and contain identical residues at the sites responsible for the observed conformation. This implies that these hypervariable regions have conformations close to those in the known structures. For five of the hypervariable regions, the repertoire of conformations appears to be limited to a relatively small number of discrete structural classes. We call the commonly occurring main-chain conformations of the hypervariable regions "canonical structures". The accuracy of the analysis is being tested and refined by the prediction of immunoglobulin structures prior to their experimental determination.     

NMR structure of the cathelin-like domain of the protegrin-3 precursor

In mammals, numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence of 94-114 residues, termed the cathelin-like domain. The cathelin-like domain of protegrin-3 (ProS) was overexpressed in Escherichia coli and uniformly labeled with (15)N or (15)N and (13)C, and its three-dimensional structure was determined by heteronuclear NMR at pH 6.2. Under these conditions and due to the cis-trans isomerization of the R(87)-P(88) and D(118)-P(119) amide bonds, the ProS structure was found to adopt four almost equally populated conformations in slow exchange on the NMR chemical shift time scale. The ProS structure consists of an N-terminal alpha-helix (Y(34)-N(48)) cradled by a four-stranded antiparallel beta-sheet (beta1, N(53)-L(60); beta2, K(74)-P(86); beta3, V(104)-V(111); and beta4, I(122)-C(124)). The solution structure of ProS, which is monomeric, allowed us to determine the structure of the L1 and L2 loops, which are too mobile in the crystal structure. The regions common to the solution and X-ray structures were found to be very similar. Finally, since the overall fold of ProS is very similar to that of cystatins despite a low degree of sequence identity, the ProS solution structure was compared to the solution and X-ray structures of the chicken cystatin. This comparison revealed that the structures of the L1 and L2 loops as well as that of the appending domain are quite different in the two proteins. These differences are mainly due to the high proline residue content (10%) which disorganizes the hydrogen bond network of a part of the ProS beta-sheet in contrast to that of the chicken cystatin structure.     

Intersegment hydrogen bonds as possible structural determinants of the N/Q/R site in glutamate receptors.

Specific electrophysiological and pharmacological properties of ionic channels in NMDA, AMPA, and kainate subtypes of ionotropic glutamate receptors (GluRs) are determined by the Asn (N), Gln (Q), and Arg (R) residues located at homologous positions of the pore-lining M2 segments (the N/Q/R site). Presumably, the N/Q/R site is located at the apex of the reentrant membrane loop and forms the narrowest constriction of the pore. Although the shorter Asn residues are expected to protrude in the pore to a lesser extent than the longer Gln residues, the effective dimension of the NMDA channel (corresponding to the size of the largest permeant organic cation) is, surprisingly, smaller than that of the AMPA channel. To explain this paradox, we propose that the N/Q/R residues form macrocyclic structures (rings) stabilized by H-bonds between a NH(2) group in the side chain of a given M2 segment and a C==O group of the main chain in the adjacent M2 segment. Using Monte Carlo minimization, we have explored conformational properties of the rings. In the Asn, but not in the Gln ring, the side-chain oxygens protruding into the pore may facilitate ion permeation and accept H-bonds from the blocking drugs. In this way, the model explains different electrophysiological and pharmacological properties of NMDA and non-NMDA GluR channels. The ring of H-bonded polar residues at the pore narrowing resembles the ring of four Thr(75) residues observed in the crystallographic structure of the KcsA K(+) channel.     

Mutations in the pore regions of the yeast K+ channel YKC1 affect gating by extracellular K+.

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wild-type current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation.     

Solution structure of omega-grammotoxin SIA,a gating modifier of P/Q and N-type Ca(2+) channel

omega-Grammotoxin SIA (GrTx) is a 36 amino acid residue protein toxin from spider venom that inhibits P/Q and N-type voltage-gated Ca(2+) channels by modifying voltage-dependent gating. We determined the three-dimensional structure of GrTx using NMR spectroscopy. The toxin adopts an "inhibitor cystine knot" motif composed of two beta-strands (Leu19-Cys21 and Cys30-Trp32) and a beta-bulge (Trp6, Gly7-Cys30) with a +2x, -1 topology, which are connected by four chain reversals. Although GrTx was originally identified as an inhibitor of voltage-gated Ca(2+) channel, it also binds to K(+) channels with lower affinity. A similar cross-reaction was observed for Hanatoxin1 (HaTx), which binds to the voltage-sensing domains of K(+) and Ca(2+) channels with different affinities. A detailed comparison of the GrTx and HaTx structures identifies a conserved face containing a large hydrophobic patch surrounded by positively charged residues. The slight differences in the surface shape, which result from the orientation of the surface aromatic residues and/or the distribution of the charged residues, may explain the differences in the binding affinity of these gating modifiers with different voltage-gated ion channels.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

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.     

Peptides and genes coding for scorpion toxins that affect ion-channels

Most scorpion toxins are ligand peptides that recognize and bind to integral membrane proteins known as ion-channels. To date there are at least 202 distinct sequences described, obtained from 30 different species of scorpions, 27 from the family Buthidae and three from the family Scorpionidae. Toxins that recognize potassium and chloride channels are usually from 29 to 41 amino acids long, stabilized by three or four disulfide bridges, whereas those that recognize sodium channels are longer, 60 to 76 amino acid residues, compacted by four disulfide bridges. Toxins specific for calcium channels are scarcely known and have variable amino acid lengths. The entire repertoire of toxins, independently of their specificity, was analyzed together by computational programs and a phylogenetic tree was built showing two separate branches. The K(+) and Cl(-) channel specific toxins are clustered into 14 subfamilies, whereas those of Na(+) and Ca(2+) specific toxins comprise at least 12 subfamilies. There are clear similarities among them, both in terms of primary sequence and the main three-dimensional folding pattern. A dense core formed by a short alpha helix segment and several antiparallel beta-sheet stretches, maintained by disulfide pairing, seems to be a common structural feature present in all toxins. The physiological function of these peptides is manifested by a blockage of ion passage through the channels or by a modification of the gating mechanism that controls opening and closing of the ion pore.     

Parallel helix bundles and ion channels: molecular modeling via simulated annealing and restrained molecular dynamics.

A parallel bundle of transmembrane (TM) alpha-helices surrounding a central pore is present in several classes of ion channel, including the nicotinic acetylcholine receptor (nAChR). We have modeled bundles of hydrophobic and of amphipathic helices using simulated annealing via restrained molecular dynamics. Bundles of Ala20 helices, with N = 4, 5, or 6 helices/bundle were generated. For all three N values the helices formed left-handed coiled coils, with pitches ranging from 160 A (N = 4) to 240 A (N = 6). Pore radius profiles revealed constrictions at residues 3, 6, 10, 13, and 17. A left-handed coiled coil and a similar pattern of pore constrictions were observed for N = 5 bundles of Leu20. In contrast, N = 5 bundles of Ile20 formed right-handed coiled coils, reflecting loosened packing of helices containing beta-branched side chains. Bundles formed by each of two classes of amphipathic helices were examined: (a) M2a, M2b, and M2c derived from sequences of M2 helices of nAChR; and (b) (LSSLLSL)3, a synthetic channel-forming peptide. Both classes of amphipathic helix formed left-handed coiled coils. For (LSSLLSL)3 the pitch of the coil increased as N increased from 4 to 6. The M2c N = 5 helix bundle is discussed in the context of possible models of the pore domain of nAChR.