The Journal of Physical Chemistry B ARTICLE Figure 1. (A) Chemical structures of glutamate and AMPA. The 3-oxyanion oxygen and nitrogen from the ring of AMPA were thought to mimic the γ- carboxylate of the glutamate. Cartoons of glutamate from the 1FTJ structure (B) and AMPA from the 1FTM structure (C) bound to GluA2 along with the water molecules that act as surrogate ligands. WG occupies the site occupied by the ring nitrogen of AMPA. WA sits in the site occupied by left-most (in this view) carboxylate oxygen of glutamate. The ionotropic glutamate receptors (iGluRs) can be classi- ﬁed, both in terms of sequence similarity and pharmacology, into three major subclasses: (S)-2-amino-3-(3-hydroxy-5-methyl-4- isoxazolyl) propionic acid (AMPA; GluA1 4), N-methyl-D- aspartic acid (NMDA; GluN1 3)) and kainic acid (GluK1 5) receptors.12 These receptors are activated by glutamate, the major excitatory neurotransmitter in the vertebrate central nervous system (CNS).13 iGluRs play key roles in various physiological processes, in particular synaptic plasticity, which is thought to be essential in memory and learning.14 Furthermore, they have been associated with abnormal neuronal activity leading to a wide range of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s chorea, amyotrophic lateral sclerosis (ALS), epi- lepsy, and ischemic stroke.14 18 Thus, pharmacological interven- tion at these neuronal receptors is a valuable therapeutic strategy. Recently, the structure of a full-length tetrameric GluA2 construct was solved to a resolution of 3.6 Å.19 However, because working with the full-length protein is diﬃcult, it has in the past been more common to perform structure work with the soluble domains; the ligand-binding core domain20 and the N-terminal domain.21 23 Currently there are over 75 crystal structures for the GluA2 ligand binding domain.24 46 These X-ray structures provide a wealth of information about the interactions of agonists, antagonists, and allosteric modulators particularly with respect to the glutamate receptor subunit GluA2. Many of these structures are of suﬃcient resolution to have reasonable conﬁdence in the positions of water molecules within the binding pocket. These observations have been supported by multiple crystal structures along with data from molecular dynamics simulations.6,47,48 Prior to the crystal structures and on the basis of pharmaco- phoric models, it was thought that glutamate and AMPA would adopt similar poses within the binding pocket. However, crystal structures revealed that AMPA adopts a slightly diﬀerent bind- ing-mode with a water molecule assuming a position that would normally be occupied by the γ-carboxyl group of glutamate (Figure 1).24 It has also been reported that docking studies that did not take into account the crystallographic waters failed to obtain the correct poses.49 This unexpected diﬀerence in binding mode raises some interesting questions: (i) What is the under- lying cause for the diﬀerence? (ii) Can the diﬀerences in the position of water molecules and binding pose of the ligand be rationalized in terms of interaction energies? (iii) Does glutamate cause polarization eﬀects that AMPA does not? (iv) Can the correct binding mode be predicted by energetic (enthalpic) considerations alone? In order to address these questions we use a quantum mechanical level treatment to accurately calculate interaction energies of both glutamate and AMPA in the two diﬀerent binding modes. Ab initio calculations have been used previously in order to account for subtype speciﬁcity between GluA1 and GluA3 where the diﬀerence in the binding pocket was a phenylalanine instead of a tyrosine.50 Here we use density functional theory (DFT) to calculate the interaction energies of components of the GluA2 binding site with AMPA and glutamate. The calculations demonstrate that interaction ener- gies alone are able to discriminate between the “correct” binding mode for glutamate but not for AMPA. We also show that the interaction energy of a water found in the glutamate-bound crystal structure (1FTJ) is more favorable than its counterpart at a diﬀerent position in the AMPA-bound crystal (WA in Figure 2B). We ﬁnd that polarization eﬀects are stronger in glutamate than in AMPA and that the diﬀerence in strain energy between alternative conformations of glutamate is very small but is over 2 kcal mol 1 for the alternative conformation of AMPA. 2. METHODS The model-systems have been assembled using the high- resolution crystallographic structures of the GluA2 ligand binding core bound with glutamate and AMPA available at the Protein Data Bank with PDB Code 1FTJ (resolution 1.9 Å) and 1FTM (resolution 1.7 Å), respectively. Since the crystal structures were composed of 3 protomers (chains A, B, and C), only one 7086 dx.doi.org/10.1021/jp200776t |J. Phys. Chem. B 2011, 115, 7085–7096