Selectivity and cooperativity of modulatory ions in a neurotransmitter receptor

Abstract

Ions play a modulatory role in many proteins. Kainate receptors, members of the ionotropic glutamate receptor family, require both monovalent anions and cations in the extracellular milieu for normal channel activity. Molecular dynamics simulations and extensive relative binding free energy calculations using thermodynamic integration were performed to elucidate the rank order of binding of monovalent cations, using x-ray crystal structures of the GluR5 kainate receptor dimers with bound cations from the alkali metal family. The simulations show good agreement with experiments and reveal that the underlying backbone structure of the binding site is one of the most rigid regions of the protein. A simplified model where the partial charge of coordinating oxygens was varied suggests that selectivity arises from the presence of two carboxylate groups. Furthermore, using a potential of mean force derived from umbrella sampling, we show that the presence of cations lower the energy barrier for anion approach and binding in the buried anion binding cavity.

Figure 1

(A) Top view and (B) side view of a cartoon representation of GluR5 (3C32) dimer with subunits in green and blue. Na⁺ and Cl⁻ ions are displayed as blue and red spheres, respectively (not to scale). (C) Closeup of one of the sodium binding sites. The oxygen of a water molecule coordinating the sodium is shown as a yellow sphere (not to scale).

Figure 2

Thermodynamic cycle used to compute relative binding free energy. In this instance, A and B are cations. A is alchemically transformed to B, in bound and unbound states, and the ΔG of transformation calculated using thermodynamic integration. The relative binding free energy ΔΔG is calculated as ΔΔG(A→B) = ΔG_bind(B) − ΔG_bind(A) = ΔG_protein(A→B) − ΔG_water(A→B).

Figure 3

(A) Protein fragments used in the reduced model are shown in orange cartoon representation; the rest of the structure, which was omitted from the model, is colored as in Fig. 1A. Refer to text and Table 3 for more details. Na⁺ is shown as blue spheres, Cl⁻ as a red sphere, and water molecules as yellow spheres (spheres not to scale). Capital letters and numbers are used to indicate helices and strands, respectively. (B) Carboxylate and carbonyl groups with partial charges. Refer to Table 3 for carboxylate partial charges q_C, q_O1, and q_O2 used in the reduced model.

Figure 4

(A) Reaction coordinate used for computing the PMF for anion binding. The reaction coordinate was chosen as the perpendicular bisector of the line segment joining the centers of the two cation binding sites (blue spheres) and passing through the anion binding site (red sphere). (B) PMF profiles for the unbinding of the Cl⁻ ion in the presence (green line) and absence (red line) of bound cations. The location of the anion- and cation binding sites with respect to the chosen reference system are marked.

Figure 5

Root mean-square fluctuation (RMSF) of Cα atoms of the Na⁺-bound GluR5 structure (PDB ID: 3C32) from a 20-ns MD simulation. The RMSF of the two subunits are shown in solid and dashed lines. Residues in the cation binding site (Glu-509, Ile-512, Asp-513) reside in a region, as marked, with low backbone movement.

Figure 6

Cartoon representation of GluR5 crystal structures with binding site residues shown in stick representation and cations as spheres. Sphere radius corresponds to approximated ionic radius. Binding sites residues from the corresponding cation-bound crystal structures (3C31, 3C32, 3C33, 3C34, and 3C45) with cations shown as spheres; Li⁺ (red), Na⁺ (blue), K⁺ (green), Rb⁺ (orange), and Cs⁺ (pink).