Molecular mechanism of ion-ion and ion-substrate coupling in the Na+-dependent leucine transporter LeuT

Abstract

Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an important role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high-resolution structure for a bacterial homolog of Na(+)/Cl(-)-dependent neurotransmitter transporters, LeuT, offers a unique opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is required to ensure substrate coupling to Na(+) and not to Li(+) or K(+) cations. Furthermore, it was found that presence of the ion in site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na(+) in the case of double ion occupancy.

FIGURE 1

Snapshot of full system of LeuT (PDB 2Q6H) embedded in a lipid membrane surrounded by water. Leucine substrate is shown in blue, bound clomipramine is shown in dark magenta, and the two sodium ions are shown in yellow. Site NA1 is the leftmost ion and NA2 is on the right.

FIGURE 2

Interaction energies between NA1/NA2/leucine and whole protein. All values are in kcal/mol, and are averages over 7000 measurements from production trajectory data. Standard error for each measurement is <0.5 kcal/mol.

FIGURE 3

Graphical representation of the data in Table 3. The data points represent the LeuT active site with double-ion occupancy combinations (Na⁺, Li⁺, and K⁺). Ion sizes are not drawn to scale. All data points are relative to the Na⁺/Na⁺ model (shown in the center). The leftmost chart shows ΔΔG°_bind data and the right shows the “Corrected” ΔΔG°_bind data. Li⁺ and K⁺ ions are labeled with text (red and blue, respectively); Na⁺ ions are unlabeled (orange). Ordering on the x axis is by ion occupancy.