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Figure 2. Hierarchical clustering of NMR-derived TPN structural conformers based on pairwise RMSD analysis. A. Heatmap and cluster analysis of the calculated pairwise RMSD along the TPN 21-amino acid alpha-carbon backbone. RMSD analysis was performed using VMD software, with the resulting 21 Ã 21 matrix (21 TPN conformers by 21 TPN amino acids) analyzed using Heatmapper (http://www.heatmapper.ca). The red asterisks denote the TPN-12 and TPN-13 conformer clusters that yield the top docking scores to Kir3.2 and are structurally similar. B. Structural alignment of TPN-1 and TPN-12 conformers, highlighting the different disulfide (Cys3âCys14) bond configuration contributing to different peptide conformations.
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Figure 3. Binding pose of TPN docked to the Kir3.2 channel. A. Surface renderings of the TPN-13 conformer (shown in orange) docked to the Kir3.2 channel (subunits A, B, C, and D color-coded). TPN-13 was docked to the Kir3.2 channel closed state (3SYA.pdb) using Cluspro 2.0. Shown are a top view (upper image) from an extracellular vantage point, and a side view (lower image) where the TPN peptide is mostly occluded from the view by Kir3.2 subunit A (magenta). B. Solid side-view rendering (upper image) of the TPN-docked Kir3.2 channel with subunit A removed to expose for viewing the docked TPN-13 conformer (in orange) within the channel vestibule. The lower image is a “sectioned†side-view rendering that exposes the location of the C-terminal TPN lysine (K21) located at the mouth of the channel pore. Also visible is the juxtaposed TPN peptide with the Kir3.2 turret domain from subunit D.
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Figure 4. TPN docking profile to mammalian Kir channels. A. Dendrogram illustrating the amino acid sequence similarity of mouse and human Kir channel isoforms. A multiple sequence alignment was performed using the Constraint-based Multiple Alignment Tool (COBALT, National Center for Biotechnology Information). The tree function was used to generate the dendrogram illustrating the clustering of Kir channel subunit isoforms into their different gene subfamilies [21]. The red asterisks denote Kir channels known to be functionally blocked by TPN. B. Top-ranked docking scores for the TPN-12 conformer to the outer vestibule of 12 different homology-modelled mouse Kir channels. The rat Kir1.1 profile previously reported is also shown for comparison, and is identical to mKir1.1. The five Kir channels with the highest docking scores are color-coded; mKir1.1 (red), mKir4.1 (yellow), mKir3.4 (blue), Kir3.2 (green), and mKir3.1 (orange). C. Comparison of maximal docking scores for the TPN–Kir channel complexes. Bars represent the mean ± SD for the top five-ranked complexes for each TPN12-docked Kir channel complex. Colored bars correspond to the Kir channels color labelled in panel B.
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Figure 5. Comparative analysis of the docked tertiapin âfootprintâ among 3 of the highest scored Kir channel outer vestibules. Diagrams for the subunit assembled Kir channel tetramer arrangements (top view) for the homology-modelled mKir1.1 (red) and mKir4.1 (yellow) channels, and the mKir3.2 (green) homo-tetrameric channel. The predicted amino acid contacts between TPN and each Kir channel subunit are listed in each diagram below, with interfacing residues indicated in yellow and residues making hydrogen bonds or salt bridge link indicated in orange. Inaccessible residues are shown in dark blue, and solvent-accessible residues not involved in the TPNâKir channel inferface are shown in light blue. TPNâKir channel Interface analysis was performed using the PISA program on the top-scored docked complexes.
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Figure 6. Surface interface and contact sites for the TPN-docked mKir4.1 channel complex. A. Surface rendering of the modelled outer vestibule of the mKir4.1 channel (top view, left; side view, right). Yellow residues depict PISA-predicted interface sites, with orange residues depicting sites with predicted H-bonds with the docked TPN peptide. For the side-view image, one of the channel subunits has been removed to expose the pore region of the vestibule. B. Surface rendering of TPN (red) docked to the outer vestibule of mKir4.1 channel (top view, left; side view, right). For the side-view image, the location of TPN K21 in the pore is exposed with one of the channel subunits removed.
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Figure 7. Validation testing for functional block of mKir4.1 channels by TPNQ. A. Electrophysiological recordings of mKir4.1 channel currents before and during application of TPNQ in Xenopus oocytes. The time-course plot shows the amplitude of membrane currents at −80 mV during 2-electrode voltage clamp recording. Indicated by the red bar, high K+ (98 mM) application evokes inward mKir4.1-mediated K+ currents. Application of either 100 nM TPNQ (green) or 1 μM BaCl2 (blue) demonstrates TPNQ insensitivity and Ba2+ sensitivity of mKir4.1 currents. Right panel: Membrane currents evoked by the voltage-ramp protocol display the inward rectification properties of the mKir4.1 channel currents in the absence and presence of either 100 nM TPNQ (green) or 1 μM BaCl2 (blue). The results are representative of 7 different oocytes. B. Positive control comparison, showing similar electrophysiological recordings of rat Kir1.1 channel currents before and during application of 100 nM TPNQ in Xenopus oocytes. High K+ (20 mM) was applied to evoke inward rKir1.1-mediated K+ currents (red bar), where application of 100 nM TPNQ (green) blocked all the Kir1.1-mediated inward currents. The results are representative of 5 different oocytes.
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Figure 8. Multiple sequence alignment of six Kir channel subunit proteins identified in a BLAST search of the Aethina tumida (A. tumida) genome. The TPN-sensitive mouse Kir3.2 channel sequence was included for comparison, where the percentages of coverage (cov) and identity (pid) are shown for each A. tumida Kir (atKir) channel subunit referenced to Kir3.2. The location of the 2 transmembrane domains (TM1 and TM2) are indicated, connected by the amino acid sequence of the the outer vestibule region that forms the receptor for TPN binding and block.
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Figure 9. Docking TPN to homology-modelled atKir channels. A. Photograph of the small hive beetle, A. tumida, courtesy of the University of Florida, Entomology and Nematology Department. B. Cladogram tree from a multiple sequence alignment depicting the pairwise similarity and associated clustering among mouse Kir channel subunit protein sequences and the six identified atKir channel subunits. The neighbor-joining tree was created using the Clustal Omega program at the European Bioinformatics Institute (EMBL-EBI) without distance corrections. C. TPN docking scores for each homology-modelled atKir channel. TPN docking scores to the TPN-sensitive mouse Kir1.1 (black dotted line) and Kir3.2 channels (green dotted line) were included for benchmark comparisons. The two atKir channels having TPN docking scores similar to Kir3.2 are XP_019865983.1 (red line) and XP_019865939.1 (yellow line). Molecular docking was performed using ZDOCK and the TPN-13 conformer as described in Methods. D. Maximal TPN docking scores from the plots in panel C are shown for comparisons in descending order. The top 5 docking scores (mean ± SD) are plotted for each homology-modelled atKir channel with comparisons to those for mKir1.1 and mKir3.2.
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Figure 10. TPN docked to a homology-modelled atKir channel. A. Sequence alignment of the outer vestibule region of TPN-sensitive mouse Kir channels and the two identified atKir channels with high TPN docking scores (see Figure 8). The turret and pore regions are indicated and highlight the variable turret sequences containing acidic residues that are necessary for electrostatic contacts in TPN binding. B. Surface renderings of the outer vestibule of the homology-modelled atKir channel XP_019865983.1. Left panel: top view where PISA-predicted TPN interface sites are indicated in yellow, and sites with predicted electrostatic contacts with the docked TPN peptide indicated in orange. Center panel: top view with the docked TPN peptide shown in red, and atKir subunit turret residues (D113) indicated. Right panel: side view of the docked TPN with one atKir channel subunit removed to expose the pore region where TPN K21 is predicted to make electrostatic contacts within the atKir channel “GYG†K+ selectivity filter.
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