Exploring the Structural Basis of Ligand-specific Activation and Antagonism of an AMPA

Receptor

Yamini PurohitDepartment of Molecular & Integrative Physiology

UIUC

Ionotropic Glutamate-gated Receptors (iGluRs)

• Ligand-gated

• Ion channels

• Mediate fast excitatory synaptic transmission in the mammalian central nervous system.

Types of iGluRs

• AMPA (-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.

Transmembrane Topology of an iGluR subunit

From: Hogner et al (2002),J. Mol. Biol.; 322(1):93-109.

GluR2 ligand-binding core: S1S2

• S1 ~114 amino acids (corresponding to residues 413-527 of native GluR2 subunit)

• S2 consists of ~143 amino acids (residues 653-796 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”.

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.

S1S2J-Ligand Complexes: “Open conformations”

S1S2J: Apo S1S2J-DNQX Complex

S1S2J-Ligand Complexes: “Closed conformations”

S1S2J-Glutamate Complex S1S2J-AMPA Complex

S1S2J-Kainate Complex Apo S1S2J

Structural features of an AMPA receptor agonist

Other AMPA receptor ligandsAMPA: a full agonist DNQX: a competitive antagonist

Kainate : a partial agonist

Molecular interactions involving the -carboxyl

groups of ligands: Conserved. (1) R 485 (S1): Hydrogen bonding through -NH groups

(2) Hydrogen bonding with backbone –NH- groups of T480 (S1)

(3) Hydrogen bond with backbone –NH- group

of S654

Agonist -carboxyl interactions: Other examples

• AMPA • Kainate

Molecular interactions involving the -amino groups of ligands: Conserved tetrahedral

arrangement of bonds.

(2) backbone carbonyl oxygen of P478 (S1)

(1) -OH of T480

in S1

(3) carboxylate oxygen of

E705 (S2)

Agonist -amino interactions: Other examples

• Kainate• AMPA

The region linking the zwitterions to the -anionic moiety forms hydrophobic interaction

with Y450: Another conserved interaction

Differences in interactions of the -anionic moiety of AMPA

agonists with the S1S2 ligand-binding core can explain the

differences in their affinities and efficacies

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

Differences in Efficacies of Glutamate and Kainate at the AMPA receptor

Differences in Affinities of Glutamate and AMPA for the AMPA receptor

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

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 -stacking interactions with Y450 .

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

Sub-site map for the GluR2 ligand binding cleft