Blastocystis AOX was modeled to the TAO crystal structure (PDB:5GN2) using the Swiss-model software (http://swissmodel.expasy.org/; Arnold et al., 2006 (link); Benkert et al., 2011 (link); Biasini et al., 2014 (link)). The protein structure of Blastocystis AOX was loaded into MOE software (Molecular Operating Environment, version 2016.08, Chemical Computing Group Inc., Montreal, Canada) for some preparatory steps to correct any structural issues. Hence, the QuickPrep panel was used to optimize the hydrogen bond network using the Protonate 3D algorithm and to perform an energy minimization on the system. AMBER99 forcefield was used in assigning correct electronic charges and protonation of amino acid residues. The 3D structure for rhodoquinol was built within MOE and energy minimized using the Amber10:EHT forcefield. A second minimization was applied using the MOPAC semi-empirical energy functions (PM3 Hamiltonian). Rhodoquinol was docked into the binding site of the AOX using the Triangle Matcher placement method with London dG scoring. Subsequently, poses resulting from the placement stage were further refined using the Induced Fit method, which allows protein flexibility upon ligand binding, improving the prediction accuracy for the interaction. Poses were then rescored using the GBVI/WSA dG scoring function and the top five best scoring poses were retained.
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