Computational protocol: Molecular Modeling of a Tandem Two Pore Domain PotassiumChannel Reveals a Putative Binding Site for General Anesthetics

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Protocol publication

[…] All protein construction calculations were performed in the Discovery Studio 3.5 software suite (Accelrys, San Diego, CA). The amino acid sequence of the K2P channel from the snail, Lymnaea stagnalis (LyTASK), was obtained from the National Center for Biotechnology (NCBI) database. A BLAST sequence search was performed using this sequence to search for sequences of high homology from those of known three-dimensional (3D) structure. The two best-scored homologous human sequences were downloaded as 3D coordinates for two forms of tandem pore potassium channels. These were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) database as the human two pore domain potassium ion channels known as TWIK-1 or K2P1 (PDB code: 3UKM) at 3.4 Å resolution and TRAAK or K2P4.1 (PDB code: 3UM7) at 3.31 Å resolution. A multiple structure alignment was performed using the SAlign algorithm to create a sequence profile based upon this structural alignment. A sequence (from LyTASK) to profile (from the SAlign templates) alignment was then performed with ClustalW so as to align the sequence of the unknown structure with those of the known structures. The Modeler module was used for assignment of coordinates for aligned amino acids, the construction of possible loops, and the initial refinement of amino acid side chains. Side chain refinement was performed on all residues with only one set of potassium ions present and both undecanes present after atom typing with CHARMm atom types and CFF charges. Molecular mechanics optimization of the entire structure was performed with free side chains in vacuo and a 10 kcal mol–1 Å–2 harmonic restraint on the α carbon protein backbone. Amino acid regions on this channel that are known to be anesthetic determinants (L159 and the sequence ILRFLT) were mapped onto the resulting structure.Additional molecular modeling was performed using a modified version of a more recently released template of a tandem pore potassium channel with a different amino acid backbone connectivity. The TRAAK or K2P4.1 channel (PDB code: 4I9W(,)) is a tandem pore potassium channel that is insensitive to anesthetics, but provides higher resolution analysis that suggests altered backbone connectivity. Even though this region of controversial connectivity is quite distant from the anesthetic binding site in question, another model of LyTASK was constructed utilizing the amino acid backbone connectivity corrected to that of 4I9W along with 3UKM as the primary template. Once again, the LyTASK sequence was aligned to this, now a mix of templates, via the ClustalW algorithm. The Modeler module was used for assignment of coordinates for aligned amino acids, the construction of possible loops, and the initial refinement of amino acid side chains. […]

Pipeline specifications

Software tools SALIGN, Clustal W, CHARMM
Application Protein structure analysis
Chemicals Amino Acids, Potassium