Computational protocol: Molecular Docking Simulations Provide Insights in the Substrate Binding Sites and Possible Substrates of the ABCC6 Transporter

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Protocol publication

[…] Initially, 8 previously reported in vitro substrates () were designed and validated to use as reference ligands to perform molecular docking. Ligand coordinates (2D structure; ) were obtained from the DrugBank , PubChem Compound database (http://pubchem.ncbi.nlm.nih.gov/) and ZINC database . Second, vitamin K1 (VK1) and vitamin K2 (VK2) were evaluated as ligands, as well as vitamin K3 (VK3). The latter was used as a negative control, as it has been previously shown not to be transported by ABCC6 . Third, after delineation of predicted binding site(s), all 4651 metabolites listed in the Human Serum Metabolome Database (HSMD; http://www.serummetabolome.ca/scripts/hmdbDownload.cgi) were used to dock our ABCC6 open conformation 3D homology model. The substrate molecules with no available coordinates were drawn and converted into 3D structure, and generation of mol-files was carried out using the ACD/ChemSketch software . Energy minimization of these compounds was then performed using the United Force field (UFF) program . [...] To identify the substrate binding sites and possible binding pattern of 8 reported substrates and three vitamin K isoforms with the ABCC6 transporter protein, molecular docking was performed using Autodock 4.2 and Autodock Vina . In this study, a rigid docking protocol was considered in order to reduce the computational cost and time in which the receptor was kept rigid and ligands were allowed to rotate. For blind docking, in order to remove the bias, the whole protein transmembrane (TM) region was covered with a grid. The size of the grid box was set to 80 Å x 80 Å x 80 Å (x, y and z) with 0.925 Å spacing between the grid points. To search the best conformation space of the ligand, the Lamarckian Genetic Algorithm was employed. During the blind docking process, 100 different conformers were generated for each ligand with a population size of 150 individuals. A maximum number of evaluation was set to 2,500,000 and maximum number of generation to 27,000, while other parameters were kept as default.A refined docking was then carried out in a specific area to optimize the results, for which the grid box size was also set to 80 Å×80 Å×80 Å (x, y and z) whereas the space between grid points was reduced to 0.55277 Å. For the docking process, the number of generation was reduced to 20 conformers for each ligand with a population size of 250 individuals. Maximum number of evaluation was set to 25,000,000 while maximum number of generation was set to 27,000. Other parameters were used as default. In both the initial blind docking and refined docking approach, selection of the final pose of ligand bound with the protein was done by giving priority to the lowest binding-energy conformation with largest binding-cluster of the total conformers.A detailed analysis of residues involved in the interaction between ligands and protein was conducted using the Discovery Studio 3.1 Visualizer software (Accelyrs Software Inc., Discovery Studio Modeling Environment, and Release 3.1.San Diego: Accelrys Software Inc., 2012).Manual inspection was employed to investigate the occurrence of mutations in substrate binding sites (SBS). To identify the number of naturally found mutations residing in SBS, the amino-acids involved in SBS were compared to the list of natural variants in ABCC6 at UniProt (http://www.uniprot.org/uniprot/O95255#section_features) and the unique mutations annotated in the Leiden Open Variation Database (LOVD) of the ABCC6 gene (http://www.ncbi.nlm.nih.gov/lovd/home.php?select_db=ABCC6). […]

Pipeline specifications

Software tools ChemSketch, AutoDock, AutoDock Vina
Databases LOVD DrugBank PubChem Compound
Applications Drug design, Protein interaction analysis
Organisms Homo sapiens
Diseases Connective Tissue Diseases, Pseudoxanthoma Elasticum
Chemicals Ethylmaleimide, Glutathione, Vitamin K 3