Journal of Molecular Biology
Structural Basis for AMPA Receptor Activation and Ligand Selectivity: Crystal Structures of Five Agonist Complexes with the GluR2 Ligand-binding Core
Introduction
Fast synaptic transmission in the mammalian central nervous system (CNS) is mediated primarily by glutamate receptor ion channels.1 Glutamate receptors (GluRs) exist as ligand-gated ionotropic receptors (iGluRs) and G-protein-coupled metabotropic receptors (mGluRs). The iGluRs are further categorised into three classes, on the basis of their sensitivity to the agonists (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), kainic acid (KA) and N-methyl-d-aspartic acid (NMDA).2 The AMPA-preferring receptors are constructed from the subunits GluR1–GluR4.3 Subunits GluR5–GluR7 and KA1-2 are activated by kainate and form the KA receptors,2 and NMDA-preferring receptors are formed by combinations of the seven subunits NR1, NR2A–NR2D, and NR3A and NR3B.1 iGluRs display a broad heterogeneity due to the number of possible subunit combinations, the expression of different splice forms,1 and post-transcriptional RNA editing.4 This diversity gives rise to different functional properties, pharmacological profiles, and biological roles.1., 5.
On the basis of experimental studies6., 7., 8., 9. and sequence analyses,10 several domains of the eukaryotic iGluR subunits have been identified. Each of the subunits (900–1400 amino acid residues) is composed of: (1) an amino-terminal domain (ATD); (2) a ligand-binding core (S1 and S2); (3) three membrane-spanning domains (M1, M2 and M3); (4) one cytoplasmic re-entrant loop (P); and (5) a C-terminal intracellular region (Figure 1(a)). The two discontinuous segments S1 and S2, responsible for binding the neurotransmitter, are related in terms of amino acid sequence and structure to the bacterial periplasmic-binding proteins.11., 12. By introducing a hydrophilic linker between the segments S1 and S2, previous studies have demonstrated the necessity and sufficiency of both segments to obtain binding affinities that are comparable with that of the full-length membrane-bound receptor.13., 14.
The development of simple and large-scale expression and purification methodologies for the GluR2-S1S2 constructs14., 15. have resulted in a number of high-resolution X-ray structures.12., 16. The structures in complex with AMPA, glutamate, kainate and 6,7-dinitro-2,3-quinoxalinedione (DNQX) have identified residues involved in ligand–protein interactions.12., 16. The ligands are bound in a cleft between two domains. Domain 1 is composed of segment S1 and the C-terminal end of segment S2. The C-terminal end of segment S1 ends in domain 2, which primarily is composed of segment S2. In addition, the comparison of the apo structure of GluR2-S1S2 with the above structures has revealed different degrees of domain closure induced by individual ligands, located potential subunit–subunit contact sites, and prepared the way for a proposed model for the mechanism of activation, deactivation and modulation of iGluRs.16 Furthermore, this structural information highlighted the significant role of domain–domain interactions in determining ligand affinity and specificity, as well as kinetic properties of the channels.
The design and functional studies of new agonists and antagonists have provided important information about the structural and conformational requirements for activation and deactivation of iGluRs (for a comprehensive review, see Bräuner-Osborne et al.17). A substantial number of these ligands has been derived using AMPA as a lead structure, and some of these are shown in Figure 2. For example, the 5-methyl group has been replaced by a variety of alkyl, aryl, or heteroaryl groups. This work has resulted in a wealth of ligands displaying a broad range of activities. However, AMPA is the only isoxazole-containing ligand for which there is a structure in complex with S1S2.16 It remains to be seen whether all AMPA agonists have the same binding modes and if differences in activities of structurally related AMPA analogues can be explained by different binding modes to the receptor.
Despite the large number of AMPA receptor agonists, very few display subtype selectivity for AMPA receptors. However, the AMPA analogue Br-HIBO has been identified as a subtype-selective agonist among AMPA receptors, differentiating the homomeric receptors GluR1o from GluR3o by a ca 70-fold difference in affinity of the racemate.18 On the basis of binding experiments, it has been found that mutation of a single amino acid residue (Tyr698) in GluR1o to the corresponding residue in GluR3o (Phe) accounts for an 18-fold decrease in affinity for Br-HIBO.19 Tyr698 is located in the binding site of S1S2 and corresponds to Tyr702 in GluR2o. The structural role of Tyr702 for binding of Br-HIBO is not fully understood.
Detailed structural information is crucial in order to understand the mechanism underlying ligand recognition, selectivity, and activity. Here, we report the X-ray structures of three AMPA receptor agonists with an isoxazole moiety in complex with GluR2-S1S2 (Figure 2): ACPA,20., 21. 2-Me-Tet-AMPA,22., 23. and Br-HIBO,24., 25. as well as the structures of ACPA and Br-HIBO in complex with the S1S2-Y702F mutant. The S1S2J protein has been characterised by binding experiments to verify the correct fold of the construct. Furthermore, we have performed electrophysiology experiments on oocytes expressing homomeric GluR2i receptors. The high-resolution structures of the complexes provide a decisive increase in the understanding of the receptor–ligand interactions as well as of conformational changes related to channel activation. The GluRs are associated with certain neurologic and psychiatric diseases, and are potential therapeutic targets for drugs.17., 26. These studies will facilitate the design of new selective AMPA receptor agonists.
Section snippets
Results
High-resolution X-ray structures have been determined of the GluR2 ligand-binding domain S1S2J16 in complex with the three isoxazole-containing agonists 2-Me-Tet-AMPA, ACPA and Br-HIBO, as well as of ACPA and Br-HIBO in complex with the S1S2-Y702F mutant (all numbering is according to the predicted mature GluR2 sequence;27 Figure 2 and Table 1, Table 2). The S1S2J-Y702F construct was developed in order to understand the selectivity of Br-HIBO between GluR1o and GluR3o (Ki is 0.17 μM and 12.0 μM
Discussion
Ionotropic glutamate receptors exhibit a complex molecular pharmacology that has been exploited by the generation of a large number of competitive full agonists, partial agonists and antagonists. While the number and diversity of glutamate receptor structure–activity studies are impressive, there has been no firm structural basis on which to ground an understanding of the basis for ligand–receptor selectivity. The five high-resolution X-ray structures reported here, combined with the
Construct design, expression, refolding and purification
The GluR2-S1S2 construct S1S2J was developed by Armstrong & Gouaux.16 Segment S1 starts at Gly390 and ends at Lys506, whereas S2 starts at Pro632 and ends at Ser775; the two segments are joined by the Gly–Thr linker. The point mutation S1S2J-Y702F was synthesised by the PCR-based method described for the QuickChange Site-directed Mutagenesis Kit (Stratagene) using the following oligonucleotides: PRM1: 5′-AAA TCC AAA GGA AAG TAT GCA TTC TTG CTG GAG TCC ACA ATG AAC-3′ and PRM2: 5′-GTT CAT TGT GGA
Acknowledgements
We thank Lotte Brehm, Stine B. Vogensen, Ulf Madsen, and Povl Krogsgaard-Larsen for generously supplying us with (S)-2-Me-Tet-AMPA, (S)-ACPA, and (S)-Br-HIBO. We are grateful to Neali Armstrong and Rich Olson for advice and helpful assistance. Jeremy R. Greenwood is thanked for discussion and guidance using computational techniques. Victor S. Lamzin is thanked for assistance with the program ARP/wARP and Willy R. Wriggers with the HINGEFIND script. The work was supported by grants from
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