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The edited crystal structure (water molecules included) of the AChBP (PDB
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The edited crystal structure (water molecules included) of the AChBP (PDB 1UW6 ) was imported into MOE and all hydrogen atoms were added to the structure with their standard geometry. The hydrogens corresponding to the crystallographic water near the pyridine nitrogen of each nicotine were manually redirected in order to obtain a model with hydrogen bonds between them and Leu102 and Met114 as described by Celie et al. After manually placing each water molecule and guiding it to form the required hydrogen bonds, a local energy minimization of the molecule was performed. The sequences of AChBP and extracellular ligand binding domains (LBDs) of the different subunits were aligned using CLUSTALW 2. We chose to do a full alignment, with a sequence format FASTA (Pearson) measuring the score type as a percentage, without end gaps. All other parameters (KTUP, window length, topdiag, pair gap, matrix, gap open, gap extension and gap distance) were set with the default definitions given in the web site. Similar results were obtained by Le Novre et al. (2002). (a) Cheatham, T. E., 3rd; Brooks, B.R.; Kollman, P.A. Curr. Protoc. Nucleic Acid Chem. 2001, Chapter 7. Unit 7 9. (b) Thompson, J. D.; Gibson, T. J.; Plewniak, F.; Jeanmougin, F.; Higgins, D. G. Nucleic Acids Res. 1997, 25, 4876. (c) Mackey, A. J.; Haystead, T. A.; Pearson, W. R. Mol. Cell. Proteomics. 2001, 1, 139. (d) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.; McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thomson, J. D.; Gibson, T. J.; Higgins, D. G. Bioinformatics 2007, 23, 2947. Higgins, D.; Thompson, J.; Gibson, T.; Thompson, J. D.; Higgins, D. G.; Gibson, T. J. Nucleic Acids Res.1994, 22, 4673. (e) Lopez, R. Services Programme and Loyd A. (1997) http://www.ebi.ac.uk/Tools/clustalw2/.
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MOE (The Molecular Operating Environment), Version 2006.08, software available from Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, Canada H3A 2R7
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77954215443
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note
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We validated the procedure by taking the coordinates of the nicotine molecule obtained from the 1UW6 crystal structure and docking it into the model of the apoprotein. As the 'receptor' we used all the crystallographic coordinates of 1UW6, with the nicotine removed. The validation protocol consisted of: (a) carrying out a fit of each one of the nicotine conformations obtained from a conformational database, (b) taking as 'receptor' the ensemble of protein and solvent atoms, (c) determining as 'site' the set of all atoms within a sphere of 4.5 centered at the nicotine molecule in the 1uw6 crystal, (d) using the ligand conformational data base obtained in the previous stage without an additional conformational search, (e) using the alpha PMI placement system and (f) calculating the interaction free energy dG by using the affinity dG scoring function. Cytisinoid-AChBP docking: for each ligand at each binding site, a molecular database with 100 docked conformations was obtained. The poses of each ligand were already ranked according to the best score (kcal/mol). For each ligand the higher S values were retrieved at each binding position. The procedure was repeated three times and the best score values were averaged.
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