PDBsum entry 3h6v

Membrane protein

PDB id: 3h6v

Contents

Protein chains

263 a.a. *

Ligands

GLU ×2

NS6 ×2

GOL ×2

SO4 ×7

DMS

Waters ×659

* Residue conservation analysis

PDB id:

3h6v

Name:

Membrane protein

Title:

Crystal structure of the iglur2 ligand-binding core (s1s2j-n754s) in complex with glutamate and ns5206 at 2.10 a resolution

Structure:

Glutamate receptor 2. Chain: a, b. Fragment: iglur2-flop ligand-binding core: unp residues 413-796. Synonym: glur-2, glur-b, glur-k2, glutamate receptor ionotropic, ampa 2, ampa-selective glutamate receptor 2. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Rat. Organism_taxid: 10116. Gene: gria2, glur2. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.10Å R-factor: 0.188 R-free: 0.228

Authors:

H.Hald,M.Gajhede,J.S.Kastrup

Key ref:

H.Hald et al. (2009). Distinct structural features of cyclothiazide are responsible for effects on peak current amplitude and desensitization kinetics at iGluR2. J Mol Biol, 391, 906-917. PubMed id: 19591837 DOI: 10.1016/j.jmb.2009.07.002

Date:

24-Apr-09 Release date: 28-Jul-09

Protein chains

P19491  (GRIA2_RAT) -  Glutamate receptor 2 from Rattus norvegicus

Seq: 883 a.a.

Struc: 263 a.a.*

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

DOI no: 10.1016/j.jmb.2009.07.002

J Mol Biol 391:906-917 (2009)

PubMed id: 19591837

Distinct structural features of cyclothiazide are responsible for effects on peak current amplitude and desensitization kinetics at iGluR2.

H.Hald, P.K.Ahring, D.B.Timmermann, T.Liljefors, M.Gajhede, J.S.Kastrup.

ABSTRACT

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission. Upon glutamate application, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors undergo rapid and almost complete desensitization that can be attenuated by positive allosteric modulators. The molecular mechanism of positive allosteric modulation has been elucidated previously by crystal structures of the ligand-binding core of iGluR2 in complex with, for example, cyclothiazide (CTZ). Here, we investigate the structure and function of CTZ and three closely related analogues NS1493, NS5206, and NS5217 at iGluR2, by X-ray crystallography and fast application patch-clamp electrophysiology. CTZ was the most efficacious and potent modulator of the four compounds on iGluR2(Q)(i) [E(max) normalized to response of glutamate: 754% (CTZ), 490% (NS1493), 399% (NS5206), and 476% (NS5217) and EC(50) in micromolar: 10 (CTZ), 26 (NS1493), 43 (NS5206), and 48 (NS5217)]. The four modulators divide into three groups according to efficacy and desensitization kinetics: (1) CTZ increases the peak current efficacy twice as much as the three analogues and nearly completely blocks receptor desensitization; (2) NS5206 and NS5217 have low efficacy and only attenuate desensitization partially; (3) NS1493 has low efficacy but nearly completely blocks receptor desensitization. A hydrophobic substituent at the 3-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system is important for compound efficacy, with the following ranking: norbornenyl (bicyclo[2.2.1]hept-2-ene)>cyclopentyl>methyl. The replacement of the norbornenyl moiety with a significantly less hydrophobic cyclopentane ring increases the flexibility of the modulator as the cyclopentane ring adopts various conformations at the iGluR2 allosteric binding site. The main structural feature responsible for a nearly complete block of desensitization is the presence of an NH hydrogen bond donor in the 4-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system, forming an anchoring hydrogen bond to Ser754. Therefore, the atom at the 4-position of CTZ seems to be a major determinant of receptor desensitization kinetics.

Selected figure(s)

Figure 1.
Fig. 1. Chemical structures of CTZ, NS1493, NS5206, and NS5217. The atom numbering for relevant atoms is shown in italics. NS1493, NS5206, and NS5217 are shown in neutral forms.

Figure 4.
Fig. 4. Binding modes of the allosteric modulators (a) CTZ, (b) NS1493, (c) NS5206, and (d) NS5217 in iGluR2 S1S2J-N754S. An F[o] − F[c] omit electron density map (green; contoured at 3 σ), a final 2F[o] − F[c] electron density map (light blue; contoured at 1 σ), and potential hydrogen-bonding interactions within 3.2 Å (broken lines) are shown. Oxygen atoms are colored red, nitrogen atoms are colored blue, and sulfur atoms are colored yellow. Water molecules are shown as red spheres. The chemical structures of the compounds are shown in Fig. 1.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 391, 906-917) copyright 2009.

Figures were selected by an automated process.