Molecular Dynamics Simulation Reveals Specific Interaction

toxins Article Molecular Dynamics Simulation Reveals Specific Interaction Sites between Scorpion Toxins and Kv1.2 Channel: Implications for Design of Highly Selective Drugs Shouli Yuan 1,2, Bin Gao 1 and Shunyi Zhu 1,* ID 1 Group of Peptide Biology and Evolution, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (S.Y.); [email protected] (B.G.) 2 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China * Correspondence: [email protected] Academic Editors: Bryan Grieg Fry and Steve Peigneur Received: 29 August 2017; Accepted: 19 October 2017; Published: 1 November 2017 Abstract: The Kv1.2 channel plays an important role in the maintenance of resting membrane potential and the regulation of the cellular excitability of neurons, whose silencing or mutations can elicit neuropathic pain or neurological diseases (e.g., epilepsy and ataxia). Scorpion venom contains a variety of peptide toxins targeting the pore region of this channel. Despite a large amount of structural and functional data currently available, their detailed interaction modes are poorly understood. In this work, we choose four Kv1.2-targeted scorpion toxins (Margatoxin, Agitoxin-2, OsK-1, and Mesomartoxin) to construct their complexes with Kv1.2 based on the experimental structure of ChTx-Kv1.2. Molecular dynamics simulation of these complexes lead to the identification of hydrophobic patches, hydrogen-bonds, and salt bridges as three essential forces mediating the interactions between this channel and the toxins, in which four Kv1.2-specific interacting amino acids (D353, Q358, V381, and T383) are identified for the first time. This discovery might help design highly selective Kv1.2-channel inhibitors by altering amino acids of these toxins binding to the four channel residues. Finally, our results provide new evidence in favor of an induced fit model between scorpion toxins and K+ channel interactions. Keywords: Kv1.2 channel; scorpion toxin; molecular dynamics simulation 1. Introduction + The voltage-gated K channel Kv1.2, encoded by KCNA2, is a transmembrane protein that is composed of four identical α-subunits, with each subunit having six transmembrane segments (S1–S6) and a membrane reentering P-loop. As shown by their experimental structure [1], S1–S4 form a voltage-sensor domain (VSD) and S5–S6 constitute a pore that selectively passes K+ ions [2]. Kv1.2 plays an important role in maintaining the resting membrane potential that enables efficient neuronal repolarization following an action potential [3]. Therefore, its loss or mutation will cause some neurogenic diseases, such as ataxia, myoclonic epilepsy, and premature death [4–7]. Given its key physiological function, Kv1.2 is frequently selected as a target by a diversity of venomous animals. In scorpions, at least six families of K+ channel toxins (α-KTxs, β-KTxs, δ-KTxs, κ-KTxs, λ-KTxs, and "-KTxs) are identified [2,8–12], in which α-KTxs are the largest source targeting this channel. These α-KTxs impair the K+ channel functions by blockage of their pore region. Due to high sequence similarity in this region between Kv1.2 and its two paralogs (i.e., Kv1.1 and Kv1.3) (Figure1A), toxins are not able to distinguish the three channel subtypes, resulting in Toxins 2017, 9, 354; doi:10.3390/toxins9110354 www.mdpi.com/journal/toxins Toxins 2017, 9, 354 2 of 19 Toxins 2017, 9, 354 2 of 18 undesiredeffects side when effects assayed when in vivo. assayed Hence,in vivoit remains. Hence, a great it remains challenge a greatto improve challenge the K tov1.2 improve selectivity the of K v1.2 selectivitythese natural of these toxins natural via toxinsengineer viaing engineering modification modification in the absence in of the detailed absence data of about detailed the datatoxin- about the toxin-channelchannel interactions. interactions. (A) (B) + Figure 1. Scorpion toxins and+ K channels. (A) The rKv1.1-rKv1.3 pore region sequences. Amino acids Figure 1. Scorpion toxins and K channels. (A) The rKv1.1-rKv1.3 pore region sequences. Amino acids conserved across the alignment are marked in yellow, and turret, pore helix, and filter are colored in conserved across the alignment are marked in yellow, and turret, pore helix, and filter are colored in green, blue, and red, respectively; (B) The α-KTxs studied in this work. Their functional sites α green,identified blue, and by red, mutational respectively; experiments (B) The are highlighted-KTxs studied with in a thisdouble work. underline. Their functional New interaction sites identifiedsites by mutationalpredicted by experiments reported molecular are highlighted dynamics with (MD) a doublesimulation underline. data are underlined New interaction once. For sites the predicted affinity by reportedof each molecular toxin, Kd dynamics (dissociation (MD) constant), simulation Ki (equilibrium data are underlined constant), once.or IC50 For (half the maximal affinity inhibitory of each toxin, Kd (dissociationconcentration) constant), (asterisks) Ki are (equilibrium shown in nanomole constant), (nM) or [8,13–17]. IC50 (half maximal inhibitory concentration) (asterisks) are shown in nanomole (nM) [8,13–17]. There is only one experimental toxin-channel complex (ChTx-Kv1.2) available currently [18], which hampers a detailed comparative study to draw commonality and difference among complexes. There is only one experimental toxin-channel complex (ChTx-K 1.2) available currently [18], An alternative approach is to employ computative technology to solve thisv question. Several popular whichmethods hampers include a detailed homology comparative modeling, study Brownian to draw dynamics, commonality molecular and differencedocking, and among molecular complexes. An alternativedynamics approachsimulation is[19]. to employMolecular computative dynamics simulation technology is to a solvepowerful this question.tool in predicting Several popularthe methodsstructures include of toxin-channel homology modeling, complexes. BrownianSome toxin-K dynamics,v1.2 complexes molecular were reported, docking, which and molecularwere dynamicsconstructed simulation with molecular [19]. Molecular dynamics dynamicssimulation, simulationsuch as maurotoxin-K is a powerfulv1.2 [20,21]. tool inHowever, predicting the the structurescomparison of toxin-channel of the mechanism complexes. among these Some Kv1.2 toxin-K inhibitorsv1.2 is complexeslacking. were reported, which were In this work, we employed molecular dynamics (MD) simulation to study the interactions of constructed with molecular dynamics simulation, such as maurotoxin-Kv1.2 [20,21]. However, the four α-KTxs (Margatoxin (abbreviated as MgTx), Agitoxin-2 (abbreviated as AgTx-2), OsK-1, and comparison of the mechanism among these Kv1.2 inhibitors is lacking. Mesomartoxin (abbreviated as MMTX) with the pore of Kv1.2. These toxins all bind to the channel In this work, we employed molecular dynamics (MD) simulation to study the interactions of with high affinity [8,13,14] (Figure 1B). However, as mentioned above, they are also ligands of Kv1.1 four α-KTxs (Margatoxin (abbreviated as MgTx), Agitoxin-2 (abbreviated as AgTx-2), OsK-1, and and Kv1.3, with the exception of MMTX, which lacks effect on Kv1.1. Our MD simulation data reveals Mesomartoxin (abbreviated as MMTX) with the pore of K 1.2. These toxins all bind to the channel for the first time four Kv1.2-specific amino acids that are involvv ed in direct interactions with these withtoxins. high affinity This finding [8,13 ,thus14] (Figure provides1B). a structural However, basis as mentionedfor their Kv1.2 above, blocking they activity are also and ligands might help of K v1.1 and Kdesignv1.3, withnew K thev1.2-targeted exception peptide of MMTX, drugs which with lacksan improved effect on channel Kv1.1. subtype Our MD selectivity. simulation data reveals for the first time four Kv1.2-specific amino acids that are involved in direct interactions with these 2. Results toxins. This finding thus provides a structural basis for their Kv1.2 blocking activity and might help design new K 1.2-targeted peptide drugs with an improved channel subtype selectivity. 2.1. The Channelv Selectivity of Four Scorpion Toxins Analyzed 2. ResultsAll the four toxins fold into the cysteine stabilized α-helix/β-sheet (CSα/β) structure with a functional dyad comprising a conserved lysine and an aromatic amino acid in a distance of 5–7 Å 2.1. The(Figure Channel 1B). SelectivityThese toxins of Fourreversibly Scorpion block Toxins K+ channels Analyzed by interacting at the external pore of the channel protein. Of them, MgTx is a 39 amino acids peptide isolated from the venom of the scorpion All the four toxins fold into the cysteine stabilized α-helix/β-sheet (CSα/β) structure with a Centruroides margaritatus, which blocks Kv1.2 and Kv1.3 at picomolar concentrations and Kv1.1 at functional dyad comprising a conserved lysine and an aromatic amino acid in a distance of 5–7 Å nanomolar concentrations without detectable effect on other types of K+ channels, such as Kv1.4–Kv1.7 + (Figureand1B). the Theseinsect Shaker toxins K+reversibly channel [15]. block K28 of K MgTxchannels is responsible by interacting for blocking at K the+ channels external [22]. pore AgTx- of the channel protein. Of them, MgTx is a 39 amino acids peptide isolated from the venom of the scorpion Centruroides margaritatus, which blocks Kv1.2 and Kv1.3 at picomolar concentrations and Kv1.1 at + nanomolar concentrations without detectable effect on other types of K channels, such as Kv1.4–Kv1.7 Toxins 2017, 9, 354 3 of 19 and the insect Shaker K+ channel [15]. K28 of MgTx is responsible for blocking K+ channels [22]. AgTx-2Toxins is a2017 38, 9 amino, 354 acids peptide isolated from the venom of Leiurus quinquestriatus hebraeus3 of, 18 which reversibly inhibits Kv1.1 to Kv1.3 with Kd values of 0.13 nM, 3.4 nM, and 0.05 nM, respectively [8,16]. Its functional2 is a 38 residuesamino acids include peptide K27 isolated and N30 from [17 the].

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