Binding site and ligand flexibility revealed by high resolution crystal structures of GluK1 competitive antagonists

Alushin, Gregory M.; Jane, David E.; Mayer, Mark L.

doi:10.1016/j.neuropharm.2010.06.002

Cited by 24 publications

(34 citation statements)

References 52 publications

(82 reference statements)

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“…For the DKAiR1D AP5 complex, after superposition using domain 1 coordinates, rotations of 30° and 23° for the A and B subunits were required to superimpose domain 2 on the closed cleft DKaiR1D glutamate complex (Figure 5D and Figures S3D and E). The 30° opening observed for the B subunit of the AP5 complex (Figure 5D) is similar to the opening observed for several vertebrate GluK1 antagonist structures (Alushin et al, 2011; Mayer et al, 2006), and much larger than for vertebrate GluN2A NMDA receptors (Figure 5D) for which D-AP5 produces only 15° opening (Jespersen et al, 2014). …”

Section: Resultssupporting

confidence: 79%

Novel Functional Properties of Drosophila CNS Glutamate Receptors

Dharkar

Han

et al. 2016

Neuron

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Summary Phylogenetic analysis reveals AMPA, kainate and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation.

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Section: Resultssupporting

confidence: 79%

Novel Functional Properties of Drosophila CNS Glutamate Receptors

Dharkar

Han

et al. 2016

Neuron

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“…5). However, although the overall displacement of the LBD in each subunit is clearly visible, we cannot resolve the change in cleft closure that occurs in response to binding of agonist compared with the antagonist bound state (29,31); likewise, the orientation of the ligand binding site in individual LBD monomers cannot be determined with certainty at the present resolution. Nevertheless, the fits provide an impression of dramatic changes in the LBD layer that occur when binding of glutamate triggers desensitization and show how the overall conformational change between the two states might be considered to resemble the winding and unwinding of the bundle of four monomers that constitute the tetrameric receptor assembly (Movie S2).…”

Section: Stabilization Of Gluk2mentioning

confidence: 78%

“…With the goal of establishing a highly populated desensitized state suitable for structural analysis we used the agonist 2S,4R-4-methylglutamate, which binds to GluK2 with 100-fold higher affinity than glutamate (28). Likewise, to stabilize an antagonist-bound resting state we introduced four-point mutations, A487T, A658S, N690S, and F704L, based on the GluK1 LBD crystal structure (29) to create a binding site for the GluK1 selective competitive antagonist LY466195, K d 38 nM. Using tryptophan fluorescence size-exclusion chromatography (30), we established that GluK2 preparations at concentrations of 1-2 mg/ mL are monodisperse in both the resting and desensitized states when stored at 4°C in a buffer containing 2 mM n-dodecyl-β-Dmaltopyranoside (DDM) and 0.3 mM cholesterol hemisuccinate ( Fig.…”

Section: Stabilization Of Gluk2mentioning

confidence: 99%

Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain

Schauder

Kuybeda

Zhang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. Here, we report the use of single-particle cryoelectron tomography to determine the structures, at ∼21 Å resolution, of full-length GluK2 kainate receptors trapped in antagonist-bound resting and agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting cross-over between the twofold symmetric amino terminal domain and a twofold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement, resulting in a separation of the four subunits by ∼25 Å. However, the amino terminal domain, transmembrane, and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the fourfold symmetric ion channel domain. From fits of the amino terminal domain and LBD domains into the density map of the desensitized state we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.cryoelectron microscopy | ion channel gating G lutamate receptor ion channels (iGluRs) are major mediators of excitatory synaptic transmission in the central nervous system. Encoded by 18 genes in humans, each of the three major iGluR subtypes, named AMPA, kainate and NMDA receptors, has unique developmental and spatial expression profiles, with diverse kinetics and ion permeability (1). In their major functional role, these membrane proteins use the binding energy of neurotransmitters to open a cation selective pore; the resulting rapid influx of sodium and calcium ions leads to action potential generation and signaling through neuronal networks. On a longer time scale, regulation of iGluR synaptic targeting plays a key role in learning and storage of memories (2).Understanding the structural basis by which iGluRs are gated by neurotransmitters is of fundamental interest to deciphering their diverse functional roles. Arranged like beads on a string, iGluR subunits contain four distinct domains, for which the extracellular amino terminal (ATD) and ligand binding (LBD) domains can be genetically isolated, expressed as soluble proteins, and crystallized as dimers (3). These domains are linked by short polypeptide linkers to the ion channel and intracellular carboxyl terminus. The 3.6-Å resolution X-ray structure of an engineered AMPA receptor construct (GluA2 cryst Protein Data Bank ID code 3KG2), trapped in the closed s...

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“…One X-ray structure of a full length iGluR (AMPA subtype) is available today (Sobolevsky et al, 2009); however, most of the structural studies of iGluRs were made on isolated soluble domains. Within the kainate receptor subfamily numerous structures of the GluK1 LBD and several structures of the GluK2 LBD have been solved in complex with agonists: among others glutamate (Mayer, 2005), kainate (Plested et al, 2008), domoic acid (Hald et al, 2007), dysiherbaine (Frydenvang et al, 2009) and antagonists, such as ATPO (Hald et al, 2007) and compounds of UBP series (Alushin et al, 2010;Mayer et al, 2006). The structural information has provided key insights into biostructural mechanisms underlying receptor activation (Hald et al, 2007;Mayer, 2005) and continuously aids the design of new potential iGluR ligands (Larsen and Bunch, 2010;.…”

Section: Introductionmentioning

confidence: 99%