Transcript Slide 1

Exploring the Structural Basis
of Ligand-specific Activation
and Antagonism of an AMPA
Receptor
Yamini Purohit
Department 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 (a-amino-3-hydroxy-5-methyl-4isoxazole 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, NR3ANR3B.
Transmembrane Topology of an
iGluR subunit
From: Hogner et al (2002),J. Mol. Biol.; 322(1):93-109.
GluR2 ligand-binding core: S1S2
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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 nonconducting, 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 ligands
AMPA: a full agonist
Kainate : a partial
agonist
DNQX: a competitive antagonist
Molecular interactions involving the a-carboxyl
groups of ligands: Conserved.
(1) R 485
(S1):
Hydrogen
bonding
through e-NH
groups
(3)
Hydrogen
bond with
backbone –
NH- group
of S654
(2)
Hydrogen
bonding
with
backbone
–NHgroups of
T480 (S1)
Agonist a-carboxyl interactions: Other
examples
• AMPA
• Kainate
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)
Agonist a-amino interactions: Other
examples
• AMPA
• Kainate
The region linking the zwitterions to the ganionic moiety forms hydrophobic interaction
with Y450: Another conserved interaction
Differences in interactions of the
g-anionic moiety of AMPA
agonists with the S1S2 ligandbinding 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 pstacking 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 T686E402 interaction
observed in the ligand
bound state
Sub-site map for the GluR2 ligand binding cleft