GLUTAMATE RECEPTOR STRUCTURAL BIOLOGY
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Mark L. Mayer, PhD, Head, Section on
Neurophysiology and Biophysics Alokesh Ghosal, PhD, Postdoctoral
Fellow Michelle Horning, PhD, Postdoctoral
Fellow Carla Glasser, BS, Technical
Specialist Tanya Molchanava, PhD, Technical
Specialist |
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Ionotropic glutamate receptors
(iGluRs) are molecular pores that mediate signal transmission at the majority
of excitatory synapses in the mammalian nervous system. The seven gene
families of ionotropic glutamate receptors (iGluRs) in humans encode 18
subunits that assemble to form three major functional families named after
the ligands first used to identify iGluR subtypes in the late 1970s: AMPA,
kainate, and NMDA. Given their essential role in normal brain function and
development along with increasing evidence that dysfunction of GluR activity
mediates several central nervous system diseases and damage during stroke,
much of our work focuses directly on analysis of GluR function at the
molecular level. Atomic-resolution structural data obtained by protein
crystallization and X-ray diffraction provide a framework in which to design
electrophysiological and biochemical experiments to define the allosteric
mechanisms underlying ligand recognition and gating of ion channel activity.
The data will allow the development of subtype-selective antagonists and
allosteric modulators with novel therapeutic applications. Crystallographic
analysis of glutamate receptor structure Mayer, Ghosal The recent crystallization of
the ligand-binding cores of AMPA, kainate, and NMDA receptor subunits, and of
a related bacterial receptor from the photosynthetic bacterium Synechocystis sp. PCC 6803, has
revealed for the first time the molecular mechanisms underlying the binding
of agonists and antagonists, providing insight into the mechanisms of
activation and desensitization. During the past year, experimental efforts in
structural biology have focused on members of the kainate receptor gene
family. We resolved the structure of a GluR6 ligand-binding core complex with
glutamate, quisqualate 2S,4R,4-methylglutamate, and kainate at resolutions of
1.93 to 1.65 Å and solved a GluR5 complex with glutamate at 2.18 Å
resolution. The
GluR6 and GluR5 complexes with glutamate showed unambiguous density for
ligand, binding pocket side chains, and surrounding water molecules.
Glutamate binds in a cavity formed at the interface between domains 1 and 2.
The top of the cavity is capped by side chains that prevent access of
extracellular solvent and ions to the bound glutamate ligand. The cavity in
GluR6, volume 255 ± 15 Å3, is substantially smaller than that for
GluR5, 305 ± 6 Å 3, but larger than for GluR2, 218 ± 4 Å3.
In total, five ordered water molecules are trapped within the GluR6
ligand-binding pocket. They are conserved in GluR5, which has one additional
water molecule in a cavity generated by replacement of N690 by the smaller
S706 side chain. Two additional amino acid substitutions, the replacement of
F704 and T710 in GluR6 by L720 and S726 in GluR5, contribute to the larger
ligand-binding pocket in GluR5. Consistent with the smaller volume of the
ligand-binding cavity in the GluR2 glutamate complex, we observed only four
trapped water molecules compared with five and six in the kainate receptor
structures, of which three play important roles in the binding of glutamate
and are conserved between AMPA and kainate receptors. When
the GluR5-selective ligands ATPA and 5-iodowillardiine are docked in the
kainate receptor ligand-binding sites by superposition on the glutamate
ligand, it is immediately obvious that steric occlusion prevents the binding
of ATPA and 5-iodowillardiine to GluR6. The replacement of N690 by S706 in
GluR5 opens up a cavity that is sufficiently large to accommodate both the tert-butyl group of ATPA and the
5-position halogen atom of 5-iodowillardiine. Binding of ATPA displaces W1,
W2, W3, and W7 and removes the hydrophobic tert-butyl group from exposure to solvent. This hydrophobic
effect is consistent with the nM affinity for GluR5 of ATPA but not for AMPA,
which has only a single methyl group at the 5-position. The halogen atom in
5-iodowillardiine projects into the same cavity as the tert-butyl group of ATPA. The 4-carbonyl oxygen atom of the willardiine’s
uracil ring projects into a different subsite in the GluR5 ligand-binding pocket
and makes a hydrogen bond with W4 but displaces W3 and W7. Strikingly, the
extent of domain closure for the GluR6 kainate complex (23.3°) was less than
that for glutamate (26.6°) but much greater than the 12.3° domain closure
observed for the GluR2 kainate complex. Functional studies by Fleck and
colleagues on GluR6 with rapid application of glutamate and kainate reveal
that kainic acid acts as a partial agonist with 50 percent of the efficacy of
glutamate. The relatively high efficacy of kainate for GluR6 is consistent
with the much greater domain closure observed in the GluR6 kainate structure
and with recent work on AMPA receptors that links agonist efficacy with
domain closure. The
binding of glutamate to iGluRs triggers a complex allosteric transition
involving rearrangements of both the ion channel and the ligand-binding core.
The stability of the agonist bond complex results from both intermolecular
contacts made by ligand with the receptor binding site and intramolecular
contacts between domains 1 and 2, which are specific to the agonist-bound,
closed-cleft conformation. Kainate receptors typically bind to agonists with
higher affinity than AMPA receptors and show slower recovery from
desensitization, suggesting that the stability of the agonist-bound complexes
is greater than for AMPA receptors. Consistent with this observation, a
comparison of the glutamate-bound structures for GluR2, GluR5, and GluR6
reveals that, in the kainate receptors, helix F in domain 2 with loop 2 makes
an extensive cluster of contacts while the coil preceding helix D in domain 1
is absent in GluR2. Hogner
A, Kastrup JS, Jin R, Liljefors T, Mayer ML, Egebjerg J, Larsen IK, Gouaux E.
Structural basis for AMPA receptor activation and ligand selectivity: crystal
structures of five agonist complexes with the GluR2 ligand-binding core. J Mol Biol 2002;322:93-109. Mayer
ML, Armstrong NA. Structure and function of glutamate receptor channels. Ann Rev Physiol 2004;66:161-181. Structural basis for
glutamate receptor gating Horning, Mayer The availability of multiple
high-resolution crystal structures and the relative simplicity of AMPA
receptor ion channel activity provides a unique opportunity to design
experiments for developing and testing hypotheses about gating mechanisms.
Such an opportunity is important, for even though crystal structures reveal
the atomic organization of protein molecules, they do so for only a few,
often just one, of the multiple conformational states through which the
protein transits through during agonist-activated gating. A key hypothesis
developed as a result of crystallographic studies posits that iGluRs assemble
as dimers of dimers and that the integrity of the dimer provides a structural
scaffold, which permits the binding energy from agonists to open the ion
channel gate or to disrupt the dimer interface that permits entry into
desensitized states. To define the mechanisms responsible for stabilization
of dimer assembly in native AMPA receptors, we targeted amino acid
substitutions to individual contacts between the adjacent ligand-binding
cores, using the GluR2 crystal structure as a guide to design mutants.
Disruption of a salt bridge, hydrogen bond network, or intermolecular van der
Waals contacts between helices D and J in adjacent ligand-binding cores
greatly accelerates desensitization. Conservation of the contacts in AMPA and
kainate receptors indicates that they are important determinants of dimer
stability and that the dimer interface is a significant structural element in
the gating mechanism of these glutamate receptor families. The experiments
that proved our hypotheses involved mutation of a pair of two-fold
symmetrical salt links across the dimer interface, which is formed by residue
E486, projecting from the exposed surface of helix D, and K493 in the domain
1 of the adjacent protomer. Truncation of the lysine side chain at position
493 to alanine resulted in a 41-fold increase in the rate of desensitization
while the rate of onset of desensitization for E486A was 10.5-fold faster. The side chains of E486, K493,
N747, and E755 and the main chain peptide bonds of L483, V484, and F491
mediate a network of hydrogen bonds across the dimer interface. The network
connects the base and middle of helix J of one subunit with helix D of the
adjacent subunit and links the pair of protomers that form a dimer near the
center of the dimer contact surface. Mutation of N747 to alanine resulted in
a 19-fold accelerated rate of desensitization. Residue I481 just upstream of
helix D forms van der Waals contacts with the side chain methyl groups of
K493 and L751; also in helix D, residue L483 forms van der Waals contacts
with a hydrophobic cluster of methyl groups from residues L748, L751, and
K752 in helix J of the adjacent protomer. Truncation of I481 to alanine
abolished activation by glutamate while, for the larger side chain, the rate
of onset of desensitization for I481V was 22-fold faster than for wild type.
Truncation of L748 to alanine also increased the rate of onset of
desensitization 12.5-fold. The results provide further evidence that the
two-fold symmetric dimer interface between pairs of GluR ligand-binding cores
serves as a key structural element that permits the energy available from
agonist binding to do work on the ion channel gate. Armstrong N, Mayer M, Gouaux
E. Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic
adjustment of agonist-induced conformational changes. Proc Natl Acad Sci USA 2003;100:5736-5741. Horning MS, Mayer ML.
Regulation of AMPA receptor gating by ligand binding core dimers. Neuron 2004;41:379-388. Jin R, Banke TG, Mayer ML,
Traynelis SF, Gouaux E. Structural basis for partial agonist action at
ionotropic glutamate receptors. Nat
Neurosci 2003;6:803-810. Jin R, Horning M, Mayer ML,
Gouaux E. Mechanism of activation and selectivity in a ligand-gated ion
channel: structural and functional studies of GluR2 and quisqualate. Biochemistry 2002;41:15635-15643. Sun Y, Olson R, Horning M,
Armstrong N, Mayer M, Gouaux E. Mechanism of glutamate receptor
desensitization. Nature 2002;417:245-253. COLLABORATORS Neali Armstrong,
PhD, Eric Gouaux, PhD, Howard Hughes Medical Institute, For further
information, contact mlm@helix.nih.gov |
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