The carboxyl side chain of GluN1-Glu781 (green sticks) interacts with the bromine atom in the bromophenyl group of NVP-AAM077 via a halogen bond. potential of ?60 mV. The bath solution contained 5 mM HEPES, 100 mM NaCl, and 0.3 mM BaCl2, at pH 7.4 Rabbit Polyclonal to Cytochrome P450 2C8/9/18/19 (adjusted with potassium hydroxide). Currents were evoked by applications of 100 values were calculated with the Cheng-Prusoff equation: = IC50/(1 + [L-glutamate]/EC50). Results Structural Study of GluN1-GluN2A LBD Complexed to NVP-AAM077. To understand the binding mode and the resulting effect on the overall protein conformation, we attempted to obtain the high-resolution structure of the isolated LBD proteins. Toward this end, we took advantage of our previously established method for effectively obtaining high-resolution structures of the GluN1-GluN2A LBD complexed to antagonists (Jespersen et al., 2014). Specifically, the crystals of the GluN1-GluN2A LBD proteins were grown in the presence of glycine and glutamate and soaked against the crystallization buffer (see positions). No apparent change was observed TP-472 in the pattern of the GluN1-GluN2A subunit arrangement. Novel Binding Mode of NVP-AAM077. The 1.6 ? resolution structure from the TP-472 current crystallographic study provided an accurate map of the compound binding site (Fig. 4; Table 1). Substitution of glutamate TP-472 with NVP-AAM077 was near complete as there is no visual evidence for the remaining glutamate density after extensive crystal soaking. Our previous studies revealed elements of the binding pocket important for recognition of D-AP5 and PPDA (Jespersen et al., 2014). The phosphono group in D-AP5 occupies the subsite, Site I, consisting of polar residues including GluN2A-Ser689, GluN2A-Thr690, and GluN2A-Tyr730, whereas the phenanthrene group in PPDA occupies the subsite, Site II, consisting of hydrophobic residues (Jespersen et al., 2014). Binding of NVP-AAM077 is mediated by a number of direct interactions, including polar interactions between the dioxoquinoxalinyl group and residues from D1 (GluN2A-Ser511, -Thr513, and -Arg518), the phosphono group and residues from Site I, and the bromophenyl group and GluN1-Glu781 at Site III (Fig. 4C). The extension of the bromophenyl group toward the GluN1-GluN2A subunit interface and the direct interaction of the bromine atom with the carboxylate group of GluN1-Glu781 at Site III is a binding mode previously unobserved. The placement of the bromophenyl group at Site III displaces three water molecules present in the glutamate-GluN1-GluN2A LBD, which are involved in water-mediated polar interactions that tether GluN1-Glu781 and GluN2A-Ser689 or GluN2A-Thr690, thereby stabilizing the intersubunit interaction in addition to the opening of the GluN2A bi-lobe (Fig. 5). The distance between the bromine atom from NVP-AAM077 and the carboxylate group of GluN1-Glu781 is 3.5 ?, which slightly deviates from the ideal distance for bond formation (suggested to be 3.37 ? for a bromineCoxygen interaction) (Auffinger et al., 2004; Sirimulla et al., 2013). We speculate that a weak bromineCoxygen interaction exists between GluN1-Glu781 and a bromine atom because the electron density for the bromophenyl group and the GluN1-Glu781 is highly ordered (Fig. 5, A and B). Other key interactions between NVP-AAM077 and the GluN1-GluN2A LBD include polar interactions between the dioxoquinoline group and GluN2A-Ser689, the amino-bromophenyl group and GluN2A-Thr690, and water-mediated hydrogen bonds involving GluN2A-Val685, -Pro686, -Gly688, and -Glu691 (W1 and W2; Fig. 4A). Open in a separate window Fig. 4. Binding site of NVP-AAM077. (A) Stereoview of the NVP-AAM077 binding site. TP-472 The binding of NVP-AAM077 (yellow sticks) is mediated by a number of polar interactions and a halogen interaction (black dashed lines). The carboxyl side chain of GluN1-Glu781 (green sticks) TP-472 interacts with the bromine atom in the bromophenyl group of NVP-AAM077 via a halogen bond. (B) The Fo-Fc omit map of NVP-AAM077 contoured at 3illustrates clear density for every chemical feature of the compound. (C) Schematic illustration of the NVP-AAM077 binding site. The phosphono group and the bromophenyl group occupy Site I and Site III, respectively, whereas no chemical group binds to Site II. Helices are.