Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms

Abstract

The x-ray structure of LeuT, a bacterial homologue of Na(+)/Cl(-)-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na(+). Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na(+) over K(+) or Li(+), the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na(+) over K(+) arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na(+) is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a "snug-fit" mechanism.

Figure 1

Molecular graphics representation of the Na⁺-coupled LeuT transporter in the explicit DMPC membrane. Chloride and Na+ ions are depicted as van der Waals spheres (green and yellow, respectively). This picture was created with PyMol.

Figure 2

Binding site organization in the LeuT. (A) Site NA1: TM1: O—Ala₂₂, Oδ—Asn₂₇, TM6: O—Thr₂₅₄, Oγ—Thr₂₅₄, TM7: Oδ—Asn₂₈₆, OT₁—L-Leu. (B) Site NA2: TM1: O—Gly₂₀, O—Val₂₃, TM8: O—Ala₃₅₁, Oγ—Thr₃₅₄, Oδ—Ser₃₅₅. (C) Organization of the double NA1-NA2 binding site.

Figure 3

Simplified models considered for analysis. (A and B). Minimal model of the binding site NA1 (A) and NA2 (B), where all ligands are allowed to move freely within a sphere of 3.5 Å radius (indicated by dashed line). The calculated free energies ΔΔG is 2.41 kcal/mol for NA1 (A) and −0.3 kcal/mol for NA2 (B). (C) Na⁺-selective toy-model of the binding site NA2 described in the text. The calculated free energy is 1.49 kcal/mol when all functional groups within the 3.5 radii are allowed to fluctuate freely, while the outside groups were frozen.

Figure 4

Typical examples of the organization and connectivity in the Na⁺ binding sites found in the protein database. (A) Distorted tetrahedral binding site (methyl-coenzyme M reductase, 1HBN). Ligands involved in ion coordination: O—Ala₅₄₄, O_γ—Thr₅₄₇, O—Thr₅₄₇, O—Pro₅₄₈; (B) Distorted trigonal bi-pyramide binding site (30S ribosomal protein S6, 2J5A). Ligands involved in ion coordination: O—Leu₈₆, O—Lys₈₇, O—Asp₈₉, O—Leu92, O—HOH₃₃; (C) Octahedral binding site (7,8-Diaminopelargonic Acid Synthase, 1MLY, Ligands involved in ion coordination: O—Val₉₆, O—Thr₉₉, O_γ—Thr₉₉, O—Pro₁₀₀, O—Leu₁₀₃, O—HOH₉₂; (D) Seven coordinating ligands, including one negative charge (galactose oxidase, 1GOF).⁷³ Ligands involved in ion coordination: O—Lys₂₉, O_δ1—Asp₃₂, O—Asn₃₄, O—Thr₃₇, O_γ—Thr₃₇, O—Ala₁₄₁, O_ɛ2—Glu₁₄₂; (E) Binding site 1 of the aspartate transporter Glt^{; 74} show 4 member motif to provide coordination for Na+ ion: O—Ser₃₄₉, O—Ile_350, O—Gly₃₅₁, O—Thr₃₅₂, the further coordination is provided by O—Thr₃₀₈ and probably by S—Met311 and O—Ile309.

Figure 5

Statistics on the average coordination of Na⁺ and K⁺ from the PDB survey.