Energetic and structural interactions between delta-dendrotoxin and a voltage-gated potassium channel

Dendrotoxin proteins isolated from Mamba snake venom block potassium channels with a high degree of specificity and selectivity. Using site-directed mutagenesis we have identified residues that constitute the functional interaction surfaces of delta-dendrotoxin and its voltage-gated potassium channel receptor. delta-Dendrotoxin uses a triangular patch formed by seven side-chains (Lys3, Tyr4, Lys6, Leu7, Pro8, Arg10, Lys26) to block K(+) currents carried by a Shaker potassium channel variant. The inhibitory surface of the toxin interacts with channel residues at Shaker positions 423, 425, 427, 431, and 449 near the pore. Amino acid mutations that interact across the toxin-channel interface were identified by mutant cycle analysis. These results constrain the possible orientation of dendrotoxin with respect to the K(+) channel structure. We propose that dendrotoxin binds near the pore entryway but does not act as a physical plug.     

New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.     

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.     

Solution structure of BmKK2, a new potassium channel blocker from the venom of chinese scorpion Buthus martensi Karsch

A natural K+ channel blocker, BmKK2 (a member of scorpion toxin subfamily alpha-KTx 14), which is composed of 31 amino acid residues and purified from the venom of the Chinese scorpion Buthus martensi Karsch, was characterized using whole-cell patch-clamp recording in rat hippocampal neurons. The three dimensional structure of BmKK2 was determined with two-dimensional NMR spectroscopy and molecular modelling techniques. In solution this toxin adopted a common alpha/beta-motif, but showed distinct local conformation in the loop between alpha-helix and beta-sheet in comparison with typical short-chain scorpion toxins (e.g., CTX and NTX). Also, the alpha helix is shorter and the beta-sheet element is smaller (each strand consisted only two residues). The unusual structural feature of BmKK2 was attributed to the shorter loop between the alpha-helix and beta-sheet and the presence of two consecutive Pro residues at position 21 and 22 in the loop. Moreover, two models of BmKK2/hKv1.3 channel and BmKK2/rSK2 channel complexes were simulated with docking calculations. The results demonstrated the existence of a alpha-mode binding between the toxin and the channels. The model of BmKK2/rSK2 channel complex exhibited favorable contacts both in electrostatic and hydrophobic, including a network of five hydrogen bonds and bigger interface containing seven pairs of inter-residue interactions. In contrast, the model of BmKK2/hKv1.3 channel complex, containing only three pairs of inter-residue interactions, exhibited poor contacts and smaller interface. The results well explained its lower activity towards Kv channel, and predicted that it may prefer a type of SK channel with a narrower entryway as its specific receptor.     

Molecular simulation of the interaction of κ-conotoxin-PVIIA with the Shaker potassium channel pore

Molecular simulation techniques were appplied to predict the interaction of the voltage-dependent Shaker potassium channel with the channel-blocking toxin kappa-conotoxin-PVIIA (PVIIA). A structural thee-dimensional model of the extracellular vestibule of the potassium channel was constructed based on structural homologies with the bacterial potassium channel Kcsa, whose structure has been solved by X-ray crystallography. The docking of the PVIIA molecule was obtained by a geometric recognition algorithm, yielding 100 possible conformations. A series of residue-residue distance restraints, predicted from mutation-cycle experiments, were used to select a small set of a plausible channel-toxin complex models among the resulting possible conformations. The four final conformations, with similar characteristics, can explain most of the single-point mutation experiments done with this system. The models of the Shaker-PVIIA interaction predict two clusters of amino acids, critical for the binding of the toxin to the channel. The first cluster is the amino acids R2, I3, Q6 and K7 that form the plug of the toxin that interacts with the entrance to the selectivity filter of the channel. The second cluster of residues, R22, F23, N24 and K25, interacts with a channel region near to the external entrance of the pore vestibule. The consistency of the obtained models and the experimental data indicate that the Shaker-PVIIA complex model is reasonable and can be used in further biological studies such as the rational design of blocking agents of potassium channels and the mutagenesis of both toxins and potassium channels.     

Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin.

Electrostatic interactions between charybdotoxin (CTX), a specific peptide pore blocker of K+ channels, and a Ca(2+)-activated K+ channel were investigated with a genetically manipulable recombinant CTX. Point mutations at certain charged residues showed only small effects on the binding affinity of the toxin molecule: Lys11, Glu12, Arg19, His21, Lys31, and Lys32. Replacement by Gln at Arg25, Lys27, or Lys34 strongly decreased the affinity of the toxin. These affinity changes were mainly due to large increases of toxin dissociation rates without much effect on association rates, as if close-range interactions between the toxin and its receptor site of the channel were disrupted. We also found that the neutralization of Lys27 to Gln removed the toxin's characteristic voltage dependence in dissociation rate. Mutation and functional mapping of charged residues revealed a molecular surface of CTX which makes direct contact with the extracellular mouth of the K+ channel.     

Designed peptide analogues of the potassium channel blocker ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35-residue polypeptide cross-linked by 3 disulfide bridges. In an effort to generate truncated peptidic analogues of this potent channel blocker, we have evaluated three analogues, one in which the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges), and two in which non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. The effect of introducing a lactam bridge in the first helix of ShK toxin (to create cyclo14/18[Lys14,Asp18]ShK) was also examined to confirm that this modification was compatible with activity. All four analogues were tested in vitro for their ability to block Kv1.3 potassium channels in Xenopus oocytes, and their solution structures were determined using 1H NMR spectroscopy. The lactam bridge in full-length ShK is well tolerated, with only a 5-fold reduction in binding to Kv1.3. The truncated and stabilized analogue was inactive, apparently due to a combination of slight deviations from the native structure and alterations to side chains required for binding. One of the peptide scaffolds was also inactive because it failed to adopt the required structure, but the other had a K(d) of 92 microM. This active peptide incorporated mimics of Lys22 and Tyr23, which are essential for activity in ShK, and an Arg residue that could mimic Arg11 or Arg24 in the native toxin. Modification of this peptide should produce a more potent, low molecular weight peptidic analogue which will be useful not only for further in vitro and in vivo studies of the effect of blocking Kv1.3, but also for mapping the interactions with the pore and vestibule of this K(+) channel that are required for potent blockade.     

Electrostatics studies and molecular dynamics simulations of a homology model of the Shaker K+ channel pore

A homology model of the pore domain of the Shaker K+ channel has been constructed using a bacterial K+ channel, KcsA, as a template structure. The model is in agreement with mutagenesis and sequence variability data. A number of structural features are conserved between the two channels, including a ring of tryptophan sidechains on the outer surface of the pore domain at the extracellular end of the helix bundle, and rings of acidic sidechains close to the extracellular mouth of the channel. One of these rings, that formed by four Asp447 sidechains at the mouth of the Shaker pore, is shown by pK(A) calculations to be incompletely ionized at neutral pH. The potential energy profile for a K+ ion moved along the central axis of the Shaker pore domain model selectivity filter reveals a shallow well, the depth of which is modulated by the ionization state of the Asp447 ring. This is more consistent with the high cation flux exhibited by the channel in its conductance value of 19 pS.     

Helical structure and packing orientation of the S2 segment in the Shaker K+ channel

Six transmembrane segments, S1-S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an alpha-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane alpha helix and that the high-impact face packs against other transmembrane segments in the functional channel.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

Solution structure of potassium channel-inhibiting scorpion toxin Lq2

Lq2 is a unique scorpion toxin. Acting from the extracellular side, Lq2 blocks the ion conduction pore in not only the voltage- and Ca2+ -activated channels, but also the inward-rectifier K+ channels. This finding argues that the three-dimensional structures of the pores in these K+ channels are similar. However, the amino acid sequences that form the external part of the pore are minimally conserved among the various classes of K+ channels. Because Lq2 can bind to all the three classes of K+ channels, we can use Lq2 as a structural probe to examine how the non-conserved pore-forming sequences are arranged in space to form similar pore structures. In the present study, we determined the three-dimensional structure of Lq2 using nuclear magnetic resonance (NMR) techniques. Lq2 consists of an alpha-helix (residues S10 to L20) and a beta-sheet, connected by an alphabeta3 loop (residues N22 to N24). The beta-sheet has two well-defined anti-parallel strands (residues G26 to M29 and residues K32 to C35), which are connected by a type I' beta-turn centered between residues N30 and K31. The N-terminal segment (residues Z1 to T8) appears to form a quasi-third strand of the beta-sheet.     

Scanning mutagenesis of a Janus-faced atracotoxin reveals a bipartite surface patch that is essential for neurotoxic function

The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory neurotoxins isolated from the venom of Australian funnel web spiders. In addition to a strikingly asymmetric distribution of charged residues, from which their name is derived, these toxins contain an extremely rare vicinal disulfide bond. To shed light on the mechanism of action of these toxins and to enhance their utility as lead compounds for insecticide development, we developed a recombinant expression system for the prototypic family member, J-ACTX-Hv1c, and mapped the key functional residues using site-directed mutagenesis. An alanine scan using a panel of 24 mutants provided the first complete map of the bioactive surface of a spider toxin and revealed that the entire J-ACTX-Hv1c pharmacophore is restricted to seven residues that form a bipartite surface patch on one face of the toxin. However, the primary pharmacophore, or hot spot, is formed by just five residues (Arg(8), Pro(9), Tyr(31), and the Cys(13)-Cys(14) vicinal disulfide). The Arg(8)-Tyr(31) diad in J-ACTX-Hv1c superimposes closely on the Lys-(Tyr/Phe) diad that is spatially conserved across a range of structurally dissimilar K(+) channel blockers, which leads us to speculate that the J-ACTXs might target an invertebrate K(+) channel.     

Computational simulations of interactions of scorpion toxins with the voltage-gated potassium ion channel

Based on a homology model of the Kv1.3 potassium channel, the recognitions of the six scorpion toxins, viz. agitoxin2, charybdotoxin, kaliotoxin, margatoxin, noxiustoxin, and Pandinus toxin, to the human Kv1.3 potassium channel have been investigated by using an approach of the Brownian dynamics (BD) simulation integrating molecular dynamics (MD) simulation. Reasonable three-dimensional structures of the toxin-channel complexes have been obtained employing BD simulations and triplet contact analyses. All of the available structures of the six scorpion toxins in the Research Collaboratory for Structural Bioinformatics Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformations of the toxin significantly affect both the molecular recognition and binding energy between the two proteins. BD simulations predicted that all the six scorpion toxins in this study use their beta-sheets to bind to the extracellular entryway of the Kv1.3 channel, which is in line with the primary clues from the electrostatic interaction calculations and mutagenesis results. Additionally, the electrostatic interaction energies between the toxins and Kv1.3 channel correlate well with the binding affinities (-logK(d)s), R(2) = 0.603, suggesting that the electrostatic interaction is a dominant component for toxin-channel binding specificity. Most importantly, recognition residues and interaction contacts for the binding were identified. Lys-27 or Lys-28, residues Arg-24 or Arg-25 in the separate six toxins, and residues Tyr-400, Asp-402, His-404, Asp-386, and Gly-380 in each subunit of the Kv1.3 potassium channel, are the key residues for the toxin-channel recognitions. This is in agreement with the mutation results. MD simulations lasting 5 ns for the individual proteins and the toxin-channel complexes in a solvated lipid bilayer environment confirmed that the toxins are flexible and the channel is not flexible in the binding. The consistency between the results of the simulations and the experimental data indicated that our three-dimensional models of the toxin-channel complex are reasonable and can be used as a guide for future biological studies, such as the rational design of the blocking agents of the Kv1.3 channel and mutagenesis in both toxins and the Kv1.3 channel. Moreover, the simulation result demonstrates that the electrostatic interaction energies combined with the distribution frequencies from BD simulations might be used as criteria in ranking the binding configuration of a scorpion toxin to the Kv1.3 channel.     

Change of pore helix conformational state upon opening of cyclic nucleotide-gated channels

The structure of the pore region of the alpha subunit of the bovine rod cyclic nucleotide-gated channel was probed using cysteine-scanning mutagenesis and hydrophilic sulfhydryl-reactive methanethiosulfonate (MTS) reagents. A region homologous to the pore helix in the X-ray crystal structure of the KcsA K(+) channel showed a helical pattern of reactivity with externally applied MTS reagents. Surprisingly, three out of four of the reactive residues, all on one face of the pore helix, only reacted with MTS reagents in the closed state. A residue on the opposite face of the helix only reacted with MTS reagents in the open state. These results indicate that the pore helix (or its surroundings) undergoes a change in conformation, perhaps involving a rotation around its long axis, that opens a gate localized to the selectivity filter of the channel.     

Simulation of the interaction between ScyTx and small conductance calcium-activated potassium channel by docking and MM-PBSA

Computational methods are employed to simulate interaction of scorpion toxin ScyTx in complex with the small conductance calcium-activated potassium channel rsk2. All of available 25 structures of ScyTx in the Protein Data Bank determined by NMR were considered for improving performance of rigid protein docking of ZDOCK. Four main binding modes were found among a large number of predicted complexes by using clustering analysis, screening with expert knowledge, energy minimization, and molecular dynamics simulations. The quality and validity of the resulting complexes were further evaluated by molecular dynamics simulations with the generalized Born solvation model and by calculation of relative binding free energies with the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) in the AMBER 7 suit of programs. The complex formed by the 22nd structure of the ScyTx and rsk2 channel was identified as the most favorable complex by using a combination of computational methods, which contain further introduction of flexibility without restraining residue side chain. From the resulted spatial structure of the ScyTx and rsk2 channel, ScyTx associates the mouth of the rsk2 channel with alpha-helix rather than beta-sheet. Structural analysis first revealed that Arg(13) played a novel and vital role of blocking the pore of the rsk2 channel, whose role is remarkably different from that of highly homologous scorpion toxin P05. Between the interfaces in the ScyTx-rsk2 complex, strong electrostatic interaction and hydrogen bonds exist between Arg(13) of ScyTx and Gly-Tyr-Gly-Asp sequential residues located in the four symmetrical chains of the pore region. Simultaneously, five hydrogen bonds between Arg(6) of ScyTx and Asp(341)(C), Val(366)(C), and Pro(367)(C), and electrostatic interaction between Arg(6) of ScyTx and Asp(364)(B) and Asp(341)(C) are also found by structural analysis. In addition, His(31) located at the C-terminal of ScyTx is surrounded by Val(342)(A), Asp(364)(A), Met(365)(A), Pro(367)(B), and Asn(366)(B) within a contact distance of 4.0 A. These simulation results are in good agreement with experimental data and can effectively explain the binding phenomena between ScyTx and the potassium channel at the level of molecular spatial structure. The consistency between results of molecular modeling and experimental data strongly suggests that our spatial structure model of the ScyTx-rsk2 complex is reasonable. Therefore, molecular docking combined with molecular dynamics simulations followed by molecular mechanics Poisson-Boltzmann surface area analysis is an attractive approach for modeling scorpion toxin-potassium channel complexes a priori for further biological studies.     

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.     

Unique interaction of scorpion toxins with the hERG channel

ERG potassium channels specify one component of the delayed rectifier in the heart and are likely to play an important functional role in other excitable cells. Compared to other K+ channels, the human ERG (hERG) channel possesses an unusually long S5-P linker that presumably forms an alpha-helix important for channel function. hERG-specific toxins bind to the outer mouth of the hERG channel. Channel residues in the middle of the S5-P linker and at the pore entrance are critical for toxin binding. One of these scorpion toxins is BeKm-1. Residues critical for BeKm-1 binding to the hERG channel are located in the alpha-helix and the following loop, whereas the "traditional" interaction surface of other short scorpion toxins is formed by residues on the beta-sheet. This unique localization of BeKm-1's interaction surface and its specific action on the hERG channel suggest a unique outer mouth structure of the hERG channel. We used the mutant cycle analysis approach to define contacts in the toxin-channel complex. This information provides critical constraints and is important for molecular modeling of the hERG pore structure.     

Mechanism of charybdotoxin block of a voltage-gated K+ channel.

Charybdotoxin block of a Shaker K+ channel was studied in Xenopus oocyte macropatches. Toxin on rate increases linearly with toxin concentration in an ionic strength-dependent fashion and is competitively diminished by tetraethylammonium. On rate is insensitive to transmembrane voltage and to K+ on the opposite side of the membrane. Conversely, toxin off rate is insensitive to toxin concentration, ionic strength, and added tetraethylammonium but is enhanced by membrane depolarization or K+ (or Na+) in the trans solution. Charge neutralization of charybdotoxin Lys27, however, renders off rate voltage insensitive. Our results argue that block of voltage-gated K+ channels results from the binding of one toxin molecule, so that Lys27 enters the pore and interacts with K+ (or Na+) in the ion conduction pathway.     

Substitution of a hydrophobic residue alters the conformational stability of Shaker K+ channels during gating and assembly.

A leucine residue at position 370 (L370) in 29-4 Shaker K+ channels resides within two overlapping sequence motifs conserved among most voltage-gated channels: the S4 segment and a leucine heptad repeat. Here we investigate the effects observed upon substitution of L370 with many other uncharged amino acid residues. We find that smaller or more hydrophilic residues produce greater alterations in both activation and inactivation gating than does substitution with other large hydrophobic residues. In addition, subunits containing less conservative substitutions at position 370 are restricted in their assembly with wild-type subunits and are unlikely to form homomultimeric channel complexes. Consistent with the idea that L370 influences the tertiary structure of these channels, the results indicate that L370 undergoes specific hydrophobic interactions during the conformational transitions of gating; similar interactions may take place during the folding, insertion, or assembly of Shaker K+ channel subunits.     

Mapping the receptor site for charybdotoxin, a pore-blocking potassium channel inhibitor.

The Shaker K+ channel belongs to a family of structurally related voltage-activated cation channels that play a central role in cellular electrical signaling. By studying multiple site-directed mutants of the Shaker K+ channel, a region that forms the binding site for a pore-blocking scorpion toxin has been identified. The region contains a sequence that is highly conserved among cloned K+ channels and may contribute to the formation of the ion conduction pore.