The Journal of Physical Chemistry B Table 1. Interaction Energies with Counterpoise Correction and Basis Set Superposition Error (BSSE) for Two Speciﬁc Water Molecules (WG and W5) in the Binding Site of 1FTJ-Glu System at Four Diﬀerent Levels of Theory ARTICLE MPWB1K/gena MPWB1K/6-31 g* MPWB1K/6-31 g** B3LYP/6-31 g** water ΔECP (kcal mol 1) BSSE ΔECP (kcal mol 1) WG 14.81 8.39 15.71 W5 12.76 7.72 13.68 a gen refers to the locally dense basis set (LDBS) approach. Table 2. Fragment Interaction Energies of Water Molecules and Ligand in Each QM Systema kcal mol 1 fragment Ws L P LP WsL WsP WsLP 1FTJ-GLU W1 W2 W3 W4 W5 WG L W1 W2 W3 W4 W5 WG L W1 W2 W3 W4 W5 WA L 0.09 1.90 0.42 0.11 0.12 4.00 0.37 0.50 0.24 0.50 0.93 0.41 0.27 1.69 0.07 2.17 0.30 1.98 3.22 4.43 5.27 9.43 13.28 3.79 9.42 5.65 10.33 5.80 1.43 2.28 13.68 4.04 3.56 11.85 57.13 3.56 61.78 83.72 52.17 45.08 24.21 6.16 47.94 61.64 11.21 2.09 0.27 9.27 49.34 4.90 0.60 1FTJ-AMPA 1FTM-AMPA 13.18 34.21 29.15 22.52 15.27 3.10 26.85 12.72 25.44 16.48 13.35 4.69 37.59 18.05 20.69 5.92 5.58 2.10 7.04 4.65 5.34 8.62 19.37 3.41 8.84 5.73 9.02 3.89 0.03 1.36 9.74 4.69 11.23 15.70 12.26 0.67 52.58 38.83 5.14 15.19 380.13 30.48 13.35 12.32 24.28 81.47 329.53 20.95 24.21 10.59 26.70 12.12 18.39 327.93 3.65 29.05 23.18 21.79 14.87 7.87 30.47 18.01 24.56 14.87 9.75 4.21 37.21 14.65 19.55 11.76 6.26 1FTM-GLU W1 0.85 1.49 10.07 8.36 0.55 10.95 1.26 W2 0.77 2.64 20.55 21.54 1.15 20.34 20.40 W3 1.59 10.60 7.82 13.62 7.08 30.77 10.70 W4 0.83 2.68 53.96 22.96 2.82 51.65 23.68 W5 2.54 3.00 5.30 5.60 11.00 7.11 12.03 WA 0.05 11.54 2.11 3.62 16.21 4.13 5.59 L 304.81 a Fragments are deﬁned as follows: WX; water molecules where X is 1-5 or A or G as described in Figure 2. Ws; all other water molecules (e.g. for the W1-Ws cell for 1FTJ-Glu, Ws would mean W2, W3, W4, W5, and WG), L; ligand (AMPA or Glu), P; protein, LP; ligand and protein, WsL; water molecules and ligand, WsP; water molecules and protein, WsLP; water molecules and ligand and protein. The QM models were assembled by considering the speciﬁc ligand of interest, with water molecules within 5 Å from the BSSE 7.70 7.22 ΔECP (kcal mol 1) 15.91 13.89 BSSE 7.61 7.16 ΔECP (kcal mol 1) 12.29 9.49 BSSE 9.26 8.74 ligand and all amino acids within a distance of 10 Å from the ligand and water molecules. In the end 23 interacting residues from the GluA2 receptor were chosen that included Glu402, Tyr450, Pro478 - Thr480, Arg485, Gly648 - Lys656, Thr686, Tyr702 - Glu705, Met708, Lys730, Tyr732 (Figure 2E shows a schematic summary of the resulting QM system). Our rationale for this selection protocol was to ensure that we had the residues of the protein interacting with the ligand (and ob- served waters) in a system that was computationally feasible. The numbering employed here is according to the mature sequence as employed by Armstrong and Gouaux (2000) for ease of comparison.24 It is important to note that residues Arg485, Thr480 and Pro478 from Domain 1 and Ser654, Thr655 and Glu705 from Domain 2 directly interact with both ligands. The side-chain of each residue was shortened to keep only those hydrogen-bonded interactions with the ligand or adjacent resi- dues. Additionally, each peptide bond was replaced with a CR-H bond to reduce the size of the QM model system. The ﬁnal QM models consisted of 357 atoms for 1FTJ-Glu and 1FTM-Glu, 358 atoms for 1FTJ-AMPA and 361 atoms for 1FTM-AMPA. As such each model has 23 residues, the ligand and 6 water molecules, except 1FTJ-AMPA, which only main- tained 5 water molecules within 5 Å from the ligand after the energy minimization step. Prior to the calculation of single point interaction energies with density functional theory, each structure was initially optimized using the ab initio Restricted Hartree Fock (RHF) method56 with the 3-21G split valence basis set.57 59 To take into account the partially constraining eﬀect of the protein environment on the active-site geometry, the side-chains were geometry optimized while the backbone was “frozen”. Also, for contiguous amino acidic residues we froze the positions of atoms involved in the peptide bond. The RHF/3-21G geometry provided structural parameters that were then used as the input in a subsequent theoretical reﬁnement step with the inclusion of electron correlation eﬀects to obtain more reliable energy. These single point density functional theory calculations were carried out with the Gaussian 0360 program. Both the B3LYP61,62 and the MPW1B9563,64 functionals were used. Ideally one would have used this level of theory to have also performed the optimiza- tions, but these calculations are simply too prohibitive at the present time. Nevertheless, this combined approach should provide a means to compare the diﬀerent possible modes of interaction. Interaction energies were calculated for a series of fragments (Table 2) that form the major components of the binding pocket (waters, protein, and ligands). The interaction energies for any two fragments can systematically be corrected for by the basis set superposition error (BSSE) using the function counterpoise method of Boys and Bernardi.65 As such, the interaction energies 7088 dx.doi.org/10.1021/jp200776t |J. Phys. Chem. B 2011, 115, 7085–7096