The Journal of Physical Chemistry B ARTICLE Figure 2. Cartoon illustrating the four model systems. (A) Glutamate in 1FTJ structure. (B) AMPA in the 1FTM structure. (C) Glutamate in the 1FTM structure. (D) AMPA in the 1FTJ structure. (E) Schematic diagram indicating the atoms of the system (taken in this case from the 1FTM with AMPA bound). Green lines indicate hydrogen bonds. Not all atoms are shown for the sake of clarity. All atoms from residues 649 to 656 are present in the calculations but not all make direct contacts with the ligand. Only the atoms CR, C and O from Gly 648 are present in the calculations but also omitted here for clarity as are all hydrogen atoms. Figure drawn with ligplot.77 monomer (i.e., chain A) was selected as the starting geometry for a molecular mechanics energy minimization. This choice was based on previous work done on GluA2 protomers.51 In total, four model systems were prepared (Figure 2): two “wildtype” where the ligand (glutamate or AMPA) is in the pose observed crystal- lographically which we refer to as 1FTJ-Glu and 1FTM-AMPA and two where the ligand is in the alternative pose (Figure 2). For glutamate in the 1FTM structure this was achieved by converting AMPA to glutamate (by matching equivalent torsion angles, deleting atoms and converting the isoxazole nitrogen to an oxygen). For AMPA in the 1FTJ structure, AMPA was ﬁtted to the C, CR, Cβ, Cγ and Cδ atoms of glutamate such that the amide of AMPA ﬁtted onto the side-chain carboxylate of glutamate. Missing side-chains of the protein were completed with the WHAT IF web interface at http://swift.cmbi.kun.nl/whatif/. All the water molecules reported in the crystal structure were included in the simulations. The system was then solvated using a box of TIP3P52 water molecules. Counterions were added to neutralize the system and water molecules were randomly replaced with cations and anions to make a 150 mM solution. The system was then minimized using the steepest descent algorithm, until it converged with a force tolerance of 100 kJ mol 1 nm 1. GRO- MACS version 3.3.153 was used in these calculation utilizing the all atom OPLS-AA force ﬁeld.54,55 This energy minimized structure was then chosen as the representative structure to assemble the quantum mechanical (QM) model. 7087 dx.doi.org/10.1021/jp200776t |J. Phys. Chem. B 2011, 115, 7085–7096