PDBsum entry 2anj

Membrane protein

PDB id: 2anj

Contents

Protein chain

262 a.a. *

Ligands

KAI

Waters ×250

* Residue conservation analysis

PDB id:

2anj

Name:

Membrane protein

Title:

Crystal structure of the glur2 ligand binding core (s1s2j-y450w) mutant in complex with the partial agonist kainic acid at 2.1 a resolution

Structure:

Glutamate receptor 2. Chain: a. Fragment: ligand binding core. Synonym: glur-2, glur-b, glur-k2, glutamate receptor ionotropic, ampa 2. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Gene: gria2, glur2. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.10Å R-factor: 0.187 R-free: 0.248

Authors:

M.M.Holm,P.Naur,B.Vestergaard,M.T.Geballe,M.Gajhede,J.S.Kastrup, S.F.Traynelis,J.Egebjerg

Key ref:

M.M.Holm et al. (2005). A binding site tyrosine shapes desensitization kinetics and agonist potency at GluR2. A mutagenic, kinetic, and crystallographic study. J Biol Chem, 280, 35469-35476. PubMed id: 16103115 DOI: 10.1074/jbc.M507800200

Date:

11-Aug-05 Release date: 30-Aug-05

Protein chain

P19491  (GRIA2_RAT) -  Glutamate receptor 2 from Rattus norvegicus

Seq: 883 a.a.

Struc: 262 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

DOI no: 10.1074/jbc.M507800200

J Biol Chem 280:35469-35476 (2005)

PubMed id: 16103115

A binding site tyrosine shapes desensitization kinetics and agonist potency at GluR2. A mutagenic, kinetic, and crystallographic study.

M.M.Holm, P.Naur, B.Vestergaard, M.T.Geballe, M.Gajhede, J.S.Kastrup, S.F.Traynelis, J.Egebjerg.

ABSTRACT

Binding of an agonist to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid (AMPA) receptor family of the glutamate receptors (GluRs) results in rapid activation of an ion channel. Continuous application results in a non-desensitizing response for agonists like kainate, whereas most other agonists, such as the endogenous agonist (S)-glutamate, induce desensitization. We demonstrate that a highly conserved tyrosine, forming a wedge between the agonist and the N-terminal part of the bi-lobed ligand-binding site, plays a key role in the receptor kinetics as well as agonist potency and selectivity. The AMPA receptor GluR2, with mutations in Tyr-450, were expressed in Xenopus laevis oocytes and characterized in a two-electrode voltage clamp setup. The mutation GluR2(Y450A) renders the receptor highly kainate selective, and rapid application of kainate to outside-out patches induced strongly desensitizing currents. When Tyr-450 was substituted with the larger tryptophan, the (S)-glutamate desensitization is attenuated with a 10-fold increase in steady-state/peak currents (19% compared with 1.9% at the wild type). Furthermore, the tryptophan mutant was introduced into the GluR2-S1S2J ligand binding core construct and co-crystallized with kainate, and the 2.1-A x-ray structure revealed a slightly more closed ligand binding core as compared with the wild-type complex. Through genetic manipulations combined with structural and electrophysiological analysis, we report that mutations in position 450 invert the potency of two central agonists while concurrently strongly shaping the agonist efficacy and the desensitization kinetics of the AMPA receptor GluR2.

Selected figure(s)

Figure 1.
FIGURE 1. Structure of ionotropic glutamate receptors and ligands. A, an individual subunit is highlighted on the left and is formed by a single polypeptide chain beginning with the N-terminal LIVBP (leucine-isoleucine-valine-binding protein)-like domain, which is followed by S1 forming the majority of domain 1 (D1) of the ligand binding core (red). The amino acid chain then transverses domain 2 (D2) of the ligand binding core and forms M1, the P-loop, and M2. Subsequently, the rest of domain 2 (D2, blue) is formed by S2, ending up in the last transmembrane -helix (M3) and the intracellular C-terminal domain. The flip/flop region lies on the back, primarily of the D1. Scissors indicate where the corresponding receptor has been cleaved during construction of the GluR2-S1S2J construct. B, structures of selected AMPA receptor agonists.

Figure 7.
FIGURE 7. Details of the structure of GluR2-S1S2J(Y450W) in complex with kainate. A, schematic representation of the overall structure of GluR2-S1S2J(Y450W) with zoom on a F[o] - F[c] omit map contoured at 3.0 for kainate (blue in D1 and green in D2). B, stereo view of the ligand binding pocket of GluR2-S1S2J(Y450W) showing the hydrogen-bonding network to kainate (black). C, comparison of the ligand binding pocket of wild-type GluR2-S1S2J (6) (PDB code 1FT0 [PDB] , black) and GluR2-S1S2J(Y450W) (blue in D1 and green in D2), both in complex with kainate. The side chain of residue 450 is depicted showing the 6° difference of the aromatic wedge. Nitrogen atoms are blue, oxygen atoms are red, and sulfur atoms are yellow. The figure was prepared using Molscript (36) and Raster3d (37).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 35469-35476) copyright 2005.

Figures were selected by the author.