The Journal of Physical Chemistry B it was crystallized with. The interaction energies in each case are essentially iso-energetic pointing to the possibility that the binding mode must be due to more than the presence of the ligand alone. One contributing factor is polarization eﬀects which do not appear to be pairwise additive for these systems. One notable example is the clearly deﬁned W5 site which inﬂuences the binding of the ligand.48 Although the interaction energies are iso-energetic for W5-WsLP, the breakdown across each subtype is diﬀerent in each case. The results here suggest that considera- tion of the binding pocket in the absence of ligand(s) may not always give the correct answer without taking into consideration the signiﬁcant eﬀects from polarization. This is a particularly important aspect in the context of drug design as there is increasing interest in being able to predict how readily water molecules in the active site are displaced.5 Docking programs often have an energetic term that accounts for the internal or strain energy of the ligand and indeed this can be a contributing factor to the binding pose. The most useful measure of this strain energy though is the comparison between binding modes and in solvent. Our calculations showed that the diﬀerences in internal energy for the glutamate conformations using the PCM solvent model are very small (0.15 kcal mol 1). Previous calculations of glutamate in solution suggest that the energy diﬀerence between two minima for glutamate in solution may be as small as 0.1 kcal mol 1 and that the conformation in the crystal structure is intermediate between these two minima.73 Thus internal strain is not likely to be a factor for dictating the pose that glutamate adopts. On the other hand, the results for AMPA suggest that internal strain could be a contributing factor. Recently, Keith et al.74 demonstrated via the use of ab initio calculations, in conjunction with a solvent model, that bioactive (as observed by X-ray crystallography) conformations of ligands are very close to conformations expected in solution, suggesting that the best docking poses would impose little conformational strain energy on the ligand (less than 0.5 kcal mol 1 in two-thirds of the cases examined). The strain for AMPA in the 1FTJ is above this level (at 2.06 kcal mol 1). Together, the results support the notion that scoring functions must incorporate terms other than interaction energies in an accurate fashion if they are to be useful.75 The results from the RESP calculations suggest that polariza- tion eﬀects can be quite signiﬁcant in the case of glutamate but less so for AMPA. The charge assignments in the region of the molecule where the environment is the same in the four systems (the amino and carboxyl groups) are in good agreement with each other suggesting that the diﬀerences observed for the glutamate side-chain group are genuine and system dependent. The RESP method is one of the standard methods for calculating charges for ligands in force ﬁelds for which parameters are not already available. These calculations are usually performed in the gas phase. The variation in these charges shown in Figure 7 demonstrates that this may potentially be a source of error if simple point charges are used to evaluate electrostatic contribu- tions in molecular mechanics or docking approaches. Large, conformationally dependent variation in charge assignment for glutamate has been observed before; Odai et al reported the variation in Mulliken charges for four distinct conformations of glutamate in solution.73 More systematic work is clearly required in this area to assess how signiﬁcant these eﬀects are in general. We also should reiterate that we selected the B3LYP/6-31G** level of theory as the best compromise between accuracy and ARTICLE computational time. Although we found that basis set super- imposition error (BSSE) was the smallest at the MPW1B95/ 6-31G** level (Table 1), it is beyond our current resource to operate at that level. 5. CONCLUSION We have shown here how various components of the binding site of GluA2 contrive to dictate the binding mode for glutamate and AMPA. Our calculations highlight that for glutamate there is very little internal strain energy for the diﬀerent binding modes and that it could readily adopt any one of those conformations. The water, W5, which could be considered part of the ligand or part of the protein, appears to be energetically favorable regard- less of the ligand present (and indeed appears slightly more favorable when AMPA is bound). Although simple interaction energies of ligands and waters could partly account for the observed glutamate binding mode, they do not appear to account for the crystallographically observed binding mode of AMPA. Although there is more internal strain energy of AMPA in the 1FTJ-AMPA system, the overall interaction energy of AMPA with the system is more favorabale (than in 1FTM). Thus, there must be other factors, which control the pose of AMPA; most likely an entropic contribution, which is not considered here. Calculations for entropic calculations are beyond the scope of the current approach (optimizations would be needed for every fragment), but is something that could be performed in future studies as computational power increases. Although interaction energies can go some way to providing insight into what factors are controlling binding, they do not provide a complete picture. There are many reasons why compounds may adopt diﬀerent binding modes within a receptor, ultimately dictated by the free energy of binding, but with many diﬀerent contributions. The problem of computing that free energy is complex because often the diﬀerence in energy that dictates diﬀerent binding poses is a very small diﬀerence between very large numbers. Thus we must strive to be as accurate as possible if we are to make progress on this front. An accurate consideration of the role of waters within the binding pocket is going to be critical. Our results suggest that for establishing trends, treatment at the B3LYP/6-31G** level is suﬃcient, but that absolute values are in general much lower than that reported for MPWB95/6-31G** for two test cases. Finally, the results also indicate that polarization eﬀects could mean that initial partial charge assignments based on gas-phase calculations may lead to problems. More work is required to assess those eﬀects in detail. Having said that, there is much work in progress in developing polarizable models for molecular mechanics,76 so there is good reason to be optimistic on that front. ’ ASSOCIATED CONTENT bS Supporting Information. Tables of the basis set super- imposition errors for the interaction energies for all water molecules in the four studied systems is available. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *Tel: þ44 (0)1865-613305. Fax: þ44 (0)1865-613238. E-mail: Philip.biggin@bioch.ox.ac.uk. 7094 dx.doi.org/10.1021/jp200776t |J. Phys. Chem. B 2011, 115, 7085–7096