1. Volume 24, Issue 9, Pages 1582-1589 (September 2016)
The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate 
Ole Kristensen, Lise Baadsgaard Kristensen, Stine Møllerud, Karla Frydenvang, Darryl S. Pickering, Jette Sandholm Kastrup 
Structure 
Volume 24, Issue 9, Pages (September 2016)
DOI: /j.str
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2. Structure 2016 24, 1582-1589DOI: (10.1016/j.str.2016.06.019)
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3. Figure 1 Structure of GluK4-LBD with Kainate
(A) Cartoon representation of GluK4-LBD with lobe D1 in light green and lobe D2 in dark green. Kainate is shown in orange stick representation. A 2Fo − Fc OMIT map carved around kainate is shown as a gray mesh contoured at 1 σ. A gray arrow indicates D1-D2 domain closure. The important anchoring residue Arg507 is shown in stick representation and stippled black lines illustrate the salt bridge to kainate.
(B) Zoom on the three-residue linker (KGT, yellow) introduced between S1 and S2. The final 2Fo − Fc electron density is shown in light gray and contoured at 1 σ.
See also Figure S4.
Structure  , DOI: ( /j.str )
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4. Figure 2
Thermal Stability and Binding Affinity of Kainate, (S)-Glutamate, and (RS)-AMPA at GluK4-LBD
(A) Thermofluor assay denaturation profiles with melting temperatures (Tm) of 39°C with (S)-glutamate (black curve) and 53°C with kainate (orange curve).
(B) Comparison of binding of kainate (gray) in GluK4-LBD with a superimposed model of (S)-glutamate (black); see Supplemental Experimental Procedures. Selected GluK4 binding-site residues are shown, with D1 residues in light green and D2 residues in dark green.
(C) Homologous competition binding of kainate at five different [3H]-kainate concentrations. Shown are means ± SEM at 16–20 kainate concentrations performed in triplicate at purified GluK4-LBD. Kd = 1.9 nM, pKd = 8.72 ± 0.17.
(D) Heterologous competition curve of (S)-glutamate (closed circles) and (RS)-AMPA (open circles) at GluK4-LBD. Shown are the normalized pooled data from three separate experiments conducted in triplicate at 16–20 ligand concentrations. (S)-Glutamate, Ki = 25 nM, pKi = 7.61 ± 0.06; (RS)-AMPA, Ki = 430 nM, pKi = 6.38 ± 0.07.
Structure  , DOI: ( /j.str )
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5. Figure 3 The Orthosteric Binding Site of GluK4
(A) The binding site of GluK4 comprises 13 residues located within 4 Å of kainate. The five residues conserved among all AMPA and kainate receptors are colored light brown. Otherwise, color coding as in Figure 1.
(B) Hydrogen bonding interactions between kainate and GluK4 residues and water molecules (red spheres) are shown as black stippled lines. View is slightly rotated compared with (A).
(C) Kainate (orange) creates a close to optimal shape complementarity with Ile670 in GluK4. The three binding-site residues differing between GluK4 (green) and GluA2 (gray) are shown in stick representation.
(D) GluK4 has characteristic features of the GluA2 binding site, including a methionine that undergoes major conformational changes and the so-called interdomain lock between lobes D1 and D2.
See also Table S1, Figures S2, and S3.
Structure  , DOI: ( /j.str )
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6. Figure 4
D1-D2 Interlobe Interactions and Dimer Interface Residues in GluK4-LBD
(A) Direct and water-mediated interactions (stippled lines) between lobe D1 and D2 residues (in stick representation) in GluK4-LBD. Water molecules are shown as red spheres. Otherwise, color coding as in Figure 1. A list of contacts is available in Table S2, and D1-D2 contacts in the AMPA receptor subunit GluA2-LBD and kainate receptor subunit GluK1-LBD are shown in Figure S5.
(B) A model of a GluK1-GluK4 heterodimer was constructed by superimposing the GluK4-LBD structure on one GluK1-LBD subunit in the GluK1-GluK1 dimer (PDB: 4E0X) and adjusting side chain conformations to avoid clash. GluK1 is shown in cyan and GluK4 in green. Ala505 and Leu765 in GluK4 are shown as green spheres and Tyr521 in GluK1 as cyan spheres. Eight other residues differing between GluK4 and GluK1 are shown as green sticks. A model of a GluK4-GluK4 homodimer can be seen in Figure S6.
Structure  , DOI: ( /j.str )
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