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.     

Structure of the BgK-Kv1.1 complex based on distance restraints identified by double mutant cycles. Molecular basis for convergent evolution of Kv1 channel blockers

A structural model of BgK, a sea anemone toxin, complexed with the S5-S6 region of Kv1.1, a voltage-gated potassium channel, was determined by flexible docking under distance restraints identified by a double mutant cycles approach. This structure provides the molecular basis for identifying the major determinants of the BgK-Kv1.1 channel interactions involving the BgK dyad residues Lys(25) and Tyr(26). These interactions are (i) electrostatic interactions between the extremity of Lys(25) side chain and carbonyl oxygen atoms of residues from the channel selectivity filter that may be strengthened by solvent exclusion provided by (ii) hydrophobic interactions involving BgK residues Tyr(26) and Phe(6) and Kv1.1 residue Tyr(379) whose side chain protrudes in the channel vestibule. In other Kv1 channel-BgK complexes, these interactions are likely to be conserved, implicating both conserved and variable residues from the channels. The data suggest that the conservation in sea anemone and scorpion potassium channel blockers of a functional dyad composed of a lysine, and a hydrophobic residue reflects their use of convergent binding solutions based on a crucial interplay between these important conserved interactions.     

Mutating a critical lysine in ShK toxin alters its binding configuration in the pore-vestibule region of the voltage-gated potassium channel,Kv1.3

The voltage-gated potassium channel in T lymphocytes, Kv1.3, an important target for immunosuppressants, is blocked by picomolar concentrations of the polypeptide ShK toxin and its analogue ShK-Dap22. ShK-Dap22 shows increased selectivity for Kv1.3, and our goal was to determine the molecular basis for this selectivity by probing the interactions of ShK and ShK-Dap22 with the pore and vestibule of Kv1.3. The free energies of interactions between toxin and channel residues were measured using mutant cycle analyses. These data, interpreted as approximate distance restraints, guided molecular dynamics simulations in which the toxins were docked with a model of Kv1.3 based on the crystal structure of the bacterial K(+)-channel KcsA. Despite the similar tertiary structures of the two ligands, the mutant cycle data imply that they make different contacts with Kv1.3, and they can be docked with the channel in configurations that are consistent with the mutant cycle data for each toxin but quite distinct from one another. ShK binds to Kv1.3 with Lys22 occupying the negatively charged pore of the channel, whereas the equivalent residue in ShK-Dap22 interacts with residues further out in the vestibule, producing a significant change in toxin orientation. The increased selectivity of ShK-Dap22 is achieved by strong interactions of Dap22 with His404 and Asp386 on Kv1.3, with only weak interactions between the channel pore and the toxin. Potent and specific blockade of Kv1.3 apparently occurs without insertion of a positively charged residue into the channel pore. Moreover, the finding that a single residue substitution alters the binding configuration emphasizes the need to obtain consistent data from multiple mutant cycle experiments in attempts to define protein interaction surfaces using these data.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Docking of mu-conotoxin GIIIA in the sodium channel outer vestibule

mu-Conotoxin GIIIA (mu-CTX) is a high-affinity ligand for the outer vestibule of selected isoforms of the voltage-gated Na(+) channel. The detailed bases for the toxin's high affinity binding and isoform selectivity are unclear. The outer vestibule is lined by four pore-forming (P) loops, each with an acidic residue near the mouth of the vestibule. mu-CTX has seven positively charged residues that may interact with these acidic P-loop residues. Using pair-wise alanine replacement of charged toxin and channel residues, in conjunction with double mutant cycle analysis, we determined coupling energies for specific interactions between each P-loop acidic residue and selected toxin residues to systematically establish quantitative restraints on the toxin orientation in the outer vestibule. Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents measured by two-electrode voltage clamp. Mutant cycle analysis revealed novel, strong, toxin-channel interactions between K9/E403, K11/D1241, K11/D1532, and R19/D1532. Experimentally determined coupling energies for interacting residue pairs provided restraints for molecular dynamics simulations of mu-CTX docking. Our simulations suggest a refined orientation of the toxin in the pore, with toxin basic side-chains playing key roles in high-affinity binding. This modeling also provides a set of testable predictions for toxin-channel interactions, hitherto not described, that may contribute to high-affinity binding and channel isoform selectivity.     

Identification of a novel pharmacophore for peptide toxins interacting with K+ channels

KappaM-conotoxin RIIIK blocks TSha1 K+ channels from trout with high affinity by interacting with the ion channel pore. As opposed to many other peptides targeting K+ channels, kappaM-RIIIK does not possess a functional dyad. In this study we combine thermodynamic mutant cycle analysis and docking calculations to derive the binding mode of kappaM-conotoxin RIIIK to the TSha1 channel. The final model reveals a novel pharmacophore, where no positively charged side chain occludes the channel pore. Instead the positive-charged residues of the toxin form a basic ring; kappaM-RIIIK is anchored to the K+ channel via electrostatic interactions of this basic ring with the loop and pore helix residues of the channel. The channel amino acid Glu-354 is likely to be a fundamental determinant of the selectivity of kappaM-RIIIK for the TSha1 channel. The Cgamma-OH of Hyp-15 is in contact with the carbonyls of the selectivity filter, disturbing the charge distribution pattern necessary for the coordination of K+ ions. This novel, experimentally based pharmacophore model proves the existence of diverse binding modes of peptidic toxins to K+ channels and underlines the role of intermolecular electrostatic interactions involving channel loop side chains in determining the selectivity of toxins for specific K+ channel types.     

Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins

Agitoxin 2 (AgTx2) is a 38-residue scorpion toxin, cross-linked by three disulfide bridges, which acts on voltage-gated K(+) (Kv) channels. Maurotoxin (MTX) is a 34-residue scorpion toxin with an uncommon four-disulfide bridge reticulation, acting on both Ca(2+)-activated and Kv channels. A 39-mer chimeric peptide, named AgTx2-MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1-5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2-MTX molecule displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7, and C4-C8, which is different from that of MTX. The 3D structure of AgTx2-MTX solved by (1)H-NMR, revealed both alpha-helical and beta-sheet structures, consistent with a common alpha/beta scaffold of scorpion toxins. Pharmacological assays of AgTx2-MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2-MTX, confirmed this result and demonstrated the participation of the N-terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N-terminal domain of MTX by the one of AgTx2 in the AgTx2-MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.     

mu-conotoxin GIIIA interactions with the voltage-gated Na(+) channel predict a clockwise arrangement of the domains

Voltage-gated Na(+) channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na(+) channel (mu1) outer vestibule and selectivity filter using mu-conotoxin GIIIA (mu-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0+/-0.1, -0.9+/-0.3, and -1.4+/-0.1 kcal/mol of coupling energy to the native toxin-channel complex, respectively. mu-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.     

Dissection of binding interactions in the complex between the anti-lysozyme antibody HyHEL-63 and its antigen

Alanine-scanning mutagenesis, X-ray crystallography, and double mutant cycles were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 and HEL. Eleven HEL residues in contact with HyHEL-63 in the crystal structure of the antigen-antibody complex, and 10 HyHEL-63 residues in contact with HEL, were individually truncated to alanine in order to determine their relative contributions to complex stabilization. The residues of HEL (Tyr20, Lys96, and Lys97) most important for binding HyHEL-63 (Delta G(mutant) - Delta G(wild type) > 3.0 kcal/mol) form a contiguous patch at the center of the surface contacted by the antibody. Hot spot residues of the antibody (Delta Delta G > 2.0 kcal/mol) are organized in two clusters that juxtapose hot spot residues of HEL, resulting in energetic complementarity across the interface. All energetically critical residues are centrally located, shielded from solvent by peripheral residues that contribute significantly less to the binding free energy. Although HEL hot spot residues Lys96 and Lys97 make similar interactions with antibody in the HyHEL-63/HEL complex, alanine substitution of Lys96 results in a nearly 100-fold greater reduction in affinity than the corresponding mutation in Lys97. To understand the basis for this marked difference, we determined the crystal structures of the HyHEL-63/HEL Lys96Ala and HyHEL-63/HEL Lys97Ala complexes to 1.80 and 1.85 A resolution, respectively. Whereas conformational changes in the proteins and differences in the solvent networks at the mutation sites appear too small to explain the observed affinity difference, superposition of free HEL in different crystal forms onto bound HEL in the wild type and mutant HyHEL-63/HEL complexes reveals that the side-chain conformation of Lys96 is very similar in the various structures, but that the Lys97 side chain displays considerable flexibility. Accordingly, a greater entropic penalty may be associated with quenching the mobility of the Lys97 than the Lys96 side chain upon complex formation, reducing binding. To further dissect the energetics of specific interactions in the HyHEL-63/HEL interface, double mutant cycles were constructed to measure the coupling of 13 amino acid pairs, 11 of which are in direct contact in the crystal structure. A large coupling energy, 3.0 kcal/mol, was found between HEL residue Lys97 and HyHEL-63 residue V(H)Asp32, which form a buried salt bridge surrounded by polar residues of the antigen. Thus, in contrast to protein folding where buried salt bridges are generally destabilizing, salt bridges in protein-protein interfaces, whose residual composition is more hydrophilic than that of protein interiors, may contribute significantly to complex stabilization.     

Structural basis of the KcsA K(+) channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method

We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K(+) channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K(+) channel. Based on properties of the AgTx2 binding site with the KcsA K(+) channel, we present a surface motif that is observed in pore-blocking toxins affecting the K(+) channel. Furthermore, we also explain the structural basis of the specificity of the K(+) channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.     

Structural refinement of the hERG1 pore and voltage‐sensing domains with ROSETTA‐membrane and molecular dynamics simulations

The hERG1 gene (Kv11.1) encodes a voltage‐gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template‐driven de‐novo design with ROSETTA‐membrane modeling using side‐chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2‐Kv2.1 chimera channels, the missing parts are modeled de‐novo. The impact of several alignments on the structure of the S4 helix in the voltage‐sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage‐sensor domain; and the topology of the extracellular S5‐pore linker compared with that established by toxin foot‐printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Nuclear magnetic resonance structural studies of a potassium channel-charybdotoxin complex

Ion channels play critical roles in signaling processes and are attractive targets for treating various diseases. Here we describe an NMR-based strategy for structural analyses of potassium channel-ligand complexes using KcsA (residues 1-132, with six mutations to impart toxin binding and to mimic the eukaryotic hERG channel). Using this approach, we determined the solution structure of KcsA in complex with the high-affinity peptide antagonist charybdotoxin. The structural data reveal how charybdotoxin binds to the closed form of KcsA and makes specific contacts with the extracellular surface of the ion channel, resulting in pore blockage. This represents the first direct structural information about an ion channel complexed to a peptide antagonist and provides an experimental framework for understanding and interpreting earlier mutational analyses. The strategy presented here overcomes many of the limitations of conventional NMR approaches to helical membrane protein structure determination and can be applied in the study of the binding of druglike molecules to this important class of proteins.     

Molecular docking of the scorpion toxin Tc1 to the structural model of the voltage-gated potassium channel Kv1.1 from human Homo sapiens

In this study, structural model of the pore loop region of the voltage-gated potassium channel Kv1.1 from human Homo sapiens was constructed based on the crystallographic structure of KcsA by structural homology. The pore loop region of Kv1.1 exhibits similar folds as that of KcsA. The structural feature of the selectivity filter of Kv1.1 is nearly identical to that of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. Molecular docking experiments of the scorpion toxin Tc1 from Tityus cambridgei to the outer vestibule of KcsA as well as Kv1.1 were subsequently performed with various initial Tc1 orientations. Tc1 was found to form the most stable complexes with these two K+ channels when the side chain of Lys14 occupies the pore of the selectivity filter through electrostatic interaction. Tc1 binds preferentially towards Kv1.1 than KcsA due to stronger hydrophobic and electrostatic interactions formed between the toxin and the selectivity filter and outer vestibule of Kv1.1. Furthermore, surface complementarity of the outer vestibules of the channels to the Tc1 spatial conformations also plays an important role in stabilizing both the Tc1/KcsA and Tc1/Kv1.1 complexes.     

Mechanisms of tetraethylammonium ion block in the KcsA potassium channel

We report results from automated docking and microscopic molecular dynamics simulations of the tetraethylammonium (TEA) complexes with KcsA. Binding modes and energies for TEA binding at the external and internal sides of the channel pore are examined utilising the linear interaction energy method. Effects of the channel ion occupancy (based on our previous results for the ion permeation mechanisms) on the binding energies are considered. Calculations show that TEA forms stable complexes at both the external and internal entrances of the selectivity filter. Furthermore, the effects of the Y82V mutation are evaluated and the results show, in agreement with experimental data, that the mutant has a significantly reduced binding affinity for TEA at the external binding site, which is attributed to stabilising hydrophobic interactions between the ligand and the tyrosines.     

Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis

Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the alphaD-beta4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on alphaE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on alphaA, alphaB, alphaC, alphaD and the loops between alphaA and alphaB, alphaD and beta5, beta6 and beta7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.     

The ionization state and the conformation of Glu-71 in the KcsA K(+) channel.

The side chain of Glu-71 of the KcsA K(+) channel, an important residue in the vicinity of the selectivity filter, was not resolved in the crystallographic structure of Doyle et al. (Doyle, D. A., J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. 1998. Science. 280:69-77). Its atomic coordinates are undetermined and its ionization state is unknown. For meaningful theoretical and computational studies of the KcsA K(+) channel, it is essential to address questions about the conformation and the ionization state of this residue in detail. In previous MD simulations in which the side chain of Glu-71 is protonated and forming a strong hydrogen bond with Asp-80 it was observed that the channel did not deviate significantly from the crystallographic structure (Bernèche, S., and B. Roux. 2000. Biophys. J. 78:2900-2917). In contrast, we show here that the structure of the selectivity filter of the KcsA channel is significantly disrupted when these side chains are fully ionized on each of the four monomers. To further resolve questions about the ionization state of Glu-71 we calculated the pK(a) value of this residue using molecular dynamics free energy simulations (MD/FES) with a fully flexible system including explicit solvent and membrane and finite-difference Poisson-Boltzmann (PB) continuum electrostatics. It is found that the pK(a) of Glu-71 is shifted by approximately +10 pK(a) units. These results strongly suggest that Glu-71 is protonated under normal conditions.     

Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling

Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG's outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG's pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG's outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.     

Electrostatic and steric contributions to block of the skeletal muscle sodium channel by mu-conotoxin.

Pore-blocking toxins are valuable probes of ion channels that underlie electrical signaling. To be effective inhibitors, they must show high affinity and specificity and prevent ion conduction. The 22-residue sea snail peptide, mu-conotoxin GIIIA, blocks the skeletal muscle sodium channel completely. Partially blocking peptides, derived by making single or paired amino acid substitutions in mu-conotoxin GIIIA, allow a novel analysis of blocking mechanisms. Replacement of one critical residue (Arg-13) yielded peptides that only partially blocked single-channel current. These derivatives, and others with simultaneous substitution of a second residue, were used to elucidate the structural basis of the toxin's blocking action. The charge at residue-13 was the most striking determinant. A positive charge was necessary, though not sufficient, for complete block. Blocking efficacy increased with increasing residue-13 side chain size, regardless of charge, suggesting a steric contribution to inhibition. Charges grouped on one side of the toxin molecule at positions 2, 12, and 14 had a weaker influence, whereas residue-16, on the opposite face of the toxin, was more influential. Most directly interpreted, the data suggest that one side of the toxin is masked by close apposition to a binding surface on the pore, whereas the other side, bearing Lys-16, is exposed to an aqueous cavity accessible to entering ions. Strong charge-dependent effects emanate from this toxin surface. In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore. This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.     

Three-dimensional Model of the Pore Form of Anthrax Protective Antigen. Structure and Biological Implications

Abstract

Although pore formation by protective antigen (PA) is critical to cell intoxication by anthrax toxin (AT), the structure of the pore form of PA (the PA63 pore) has not been determined. Hence, in this study, the PA63 pore was modeled using the X-ray structures of monomeric PA and heptameric α-hemolysin (α-HL) as templates. The PA63 pore model consists of two weakly associated domains, namely the cap and stem domains. The ring-like cap domain has a length of 80 Å and an outside diameter of 120 Å, while the cylinder-like stem domain has a length of 100 Å and outside diameter of ～28 Å. This provides the PA63 pore model with a length of 180 Å. Based on experimental results, the channel in the PA63 pore model was built to have a minimum diameter of ～12 Å, depending on side chain conformations. Because of its large size and structural complexity, the all-atom model of the PA63 pore is the end-stage construction of four separate modeling projects described herein. The final model is consistent with published experimental results, including mutational analysis and channel conductance experiments. In addition, the model was energetically and hydropathically refined to optimize molecular packing within the protomers and at the protomer-protomer interfaces. By providing atomic detail to biochemical and biophysical data, the PA63 pore model may afford new insights into the binding mode of PA on the membrane surface, the prepore-pore transition, and the mechanism of cell entry by anthrax toxin.