1. Yamini Purohit Department of Molecular & Integrative Physiology UIUC
Exploring the Structural Basis of Ligand-specific Activation and Antagonism of an AMPA Receptor
Yamini Purohit
Department of Molecular & Integrative Physiology
UIUC

2. Ionotropic Glutamate-gated Receptors (iGluRs)
Ligand-gated
Ion channels
Mediate fast excitatory synaptic transmission in the mammalian central nervous system.

3. Types of iGluRs
AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-preferring: Composed of subunits GluR1- GluR4.
Kainate-preferring: Composed of subunits GluR5-GluR7 and KA1-KA2.
NMDA (the N-methyl-D-aspartate-) preferring: NR1, NR2A-NR2D, NR3A-NR3B.

4. Transmembrane Topology of an iGluR subunit
From: Hogner et al (2002),J. Mol. Biol.; 322(1):

5. GluR2 ligand-binding core: S1S2
S1 ~114 amino acids (corresponding to residues of native GluR2 subunit)
S2 consists of ~143 amino acids (residues of native GluR2)
A hydrophilic Ser-Thr linker joins S1-S2 in S1S2J.
GluR2 S1S2J- a soluble protein: Crystal structures in complex with ligands are available.
Proven to be “necessary and sufficient to bind agonists with affinities comparable with that of the native AMPA receptor”.

6. Objectives
To explore the structure of the AMPA receptor ligand-binding core (GluR2S1S2J) at the atomic level.
To explore the structural features of the ligand molecules complementary to the ligand-binding cleft of GluR2S1S2J, and the molecular interactions underlying ligand-receptor binding.
To understand difference between apo- and ligand-bound state of GluR2S1S2J, and how these differences relate to the different functional states of the receptor (for example, Shut and non-conducting, Open and conducting).
To understand the interactions that account for the differences in the affinities and efficacies of agonists.
To explore the receptor-ligand interactions differentiating an agonist of GluR2 from an antagonist.

7. S1S2J-Ligand Complexes: “Open conformations”
S1S2J: Apo
S1S2J-DNQX Complex

8. S1S2J-Ligand Complexes: “Closed conformations”
S1S2J-Glutamate Complex
S1S2J-AMPA Complex
S1S2J-Kainate Complex
Apo S1S2J

9. Structural features of an AMPA receptor agonist

10. Other AMPA receptor ligands
AMPA: a full agonist
DNQX: a competitive antagonist
Kainate : a partial agonist

11. Molecular interactions involving the a-carboxyl groups of ligands: Conserved.
(2) Hydrogen bonding with backbone –NH- groups of T480 (S1)
(1) R 485 (S1): Hydrogen bonding through e-NH groups
(3) Hydrogen bond with backbone –NH- group of S654

12. Agonist a-carboxyl interactions: Other examples
AMPA
Kainate

13. Molecular interactions involving the a-amino groups of ligands: Conserved tetrahedral arrangement of bonds.
(1) -OH of T480 in S1
(2) backbone carbonyl oxygen of P478 (S1)
(3) carboxylate oxygen of E705 (S2)

14. Agonist a-amino interactions: Other examples
AMPA
Kainate

15. The region linking the zwitterions to the g-anionic moiety forms hydrophobic interaction with Y450: Another conserved interaction

16. Differences in interactions of the g-anionic moiety of AMPA agonists with the S1S2 ligand-binding core can explain the differences in their affinities and efficacies

17. Differences in Efficacies of Glutamate and Kainate at the AMPA receptor
-Backbone –NH- of S654 and T655
-OH of T655
solvent/water mediated interactions with L650, L703 and the backbone –NH of E705

18. Differences in Efficacies of Glutamate and Kainate at the AMPA receptor

20. Differences in Affinities of Glutamate and AMPA for the AMPA receptor

21. 5-methyl group of AMPA binds in a hydrophobic pocket in domain 1, constituted of M708, and the side-chain atoms of P478 and Y405

22. Molecular interactions in DNQX binding
(1) The two –C=O groups of DNQX, form H-bonds with –NH groups of R485, and the –OH as well as the backbone –NH of T480.
(2) Quinoxalinedione ring maximizes p-stacking interactions with Y450 .

23. Molecular interactions in DNQX binding
(3) The amide nitrogen hydrogen bonds with the backbone –C=O of P478.
(4) The 6-nitro moiety interacts with Y732, Y707 and a water molecule in the binding cleft.
(5) The 7-nitro moiety is likely to hydrogen bond to –OH of T686, and prevents the T686-E402 interaction observed in the ligand bound state

25. Sub-site map for the GluR2 ligand binding cleft