Computational protocol: Mechanistic Insight into Human ether-à-go-go-related Gene (hERG) K+ Channel Deactivation Gating from the Solution Structure of the EAG Domain

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Protocol publication

[…] The EAG domain (residues 1–135) was subcloned into an Escherichia coli expression vector with a C terminus hexahistidine tag as described previously (). All NMR experiments were performed on Bruker DRX or AvanceII spectrometers operating at either 600 or 800 MHz that were fitted with cryogenically cooled probe heads. Experiments used 350-μl samples of the uniformly 15N- or 13C/15N-labeled EAG domain (0.5 mm) dissolved in 20 mm sodium acetate buffer, pH 5.0, containing 0.5 mm DTT, 0.5 mm EDTA, 0.2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.02% (w/v) sodium azide, at 25 °C. The samples were prepared in 95% H2O and 5% D2O. Detailed descriptions of the multidimensional experiments used to obtain the near complete sequence-specific 1H, 13C, and 15N assignments have been reported previously (). Additionally, 15N-NOESY-HSQC () and 13C-NOESY-HSQC () spectra were acquired with an NOE mixing period of 100 ms. These spectra were recorded over ∼68 h, with acquisition times in the indirect dimensions of ∼16 ms for 1H and ∼7 and ∼11 ms for 13C and 15N, respectively, and for 80 ms in the real time domain. Water suppression was achieved using the gradient-based WATERGATE method (). Two-dimensional 15N/1H-HSQC spectra were also acquired from a sample of the 15N-labeled EAG domain dissolved in D2O to identify any slowly exchanging amide protons in the protein (). All NMR data were processed essentially as described previously () using Topspin (Bruker Biospin Ltd.), and the resulting spectra were analyzed using the program Sparky (T. D. Goddard and D. G. Kneller, Sparky 3, University of California, San Francisco). [...] The family of converged structures was determined using the program CYANA (), as described previously (, ). Hydrogen bond constraints, involving 50 residues with slowly exchanging backbone amide signals and where the hydrogen bond acceptor was unambiguous in preliminary structures, were added to the final round of calculations. Backbone torsion angle constraints derived from the protein backbone dihedral angle prediction program TALOS+ () were included in both stages of the calculation. The 82 structures with no restraint violations (NOE violations <0.5 Å and <5° for dihedral angles) were refined with two cycles of restrained molecular dynamics simulated annealing with the AMBER 10 package () using the following protocol. Structures were initially energy-minimized (2000 steps), followed by 20 ps of simulated annealing in vacuum and then by 20 ps of simulated annealing using a generalized Born solvent model (). The system was heated to 1200 K over the 1st ps, annealed at a constant temperature for 5 ps, and then cooled to 0 K during the remaining 14 ps. Force constants were 30 kcal mol−1 Å2 for distance constraints and 1000 kcal mol−1 rad−2 for dihedral angle constraints. The 20 structures with the lowest AMBER energy and with no distance constraint violation greater than 0.24 Å were selected. Analysis of the final family of structures obtained was carried out using PROCHECK-NMR () and MOLMOL (). [...] Homology models of the cNBD and C-linker of hERG were built using Modeler (), with the HCN2 crystal structure as a template (Protein Data Bank code 1Q5O) (). 2000 initial docking configurations for the N-terminal domain bound to the cNBD were generated using ZDOCK () for each of the 82 elucidated NMR conformers. The ZDOCK output was then re-scored and ranked using ZRANK (). The top 10 complexes for each NMR conformer were saved for cluster analysis. Clustering of the 710 docked complexes was performed using the single linkage method in g_cluster, which is part of GROMACS molecular dynamics suite ().To optimize the interactions between NT1–10 and the cNBD C-helix acidic patch, Modeler was used to apply distance restraints between the Arg4/Arg5/His7 and Asp843/Glu847/Asp850/Glu857 clusters, for a set of docked solutions that bound to the side of the cNBD. An optimal model was then selected from this group based on good agreement with the known interaction data. To test the validity of this model as a docked conformation of the EAG domain, ZDOCK was used to re-dock the modeled structure onto the cNBD. […]

Pipeline specifications

Software tools Sparky, TALOS+, AMBER, PROCHECK, MOLMOL
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Homo sapiens