Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation

Abstract

The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na(+); later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule approximately 11 A above the bound leucine and 2 Na(+). We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na(+)-coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-beta-d-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 A the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na(+)-coupled symport.

Fig. 1.

n-Octyl-β-d-glucopyranoside impairs binding of leucine to the S2 site. (A) Overview of LeuT with important structural elements highlighted. (B and C) Time course of 100 nM ³H-Leu binding to LeuT-WT^OG (red open squares), -WT^DDM (red filled squares), -L400C^DDM (green filled triangles), and -L400C^OG (green open triangles). Images represent representative parallel experiments with SD of triplicates. (D and E) Stoichiometry of ³H-Leu binding by LeuT-WT^DDM (red filled squares), -WT^OG (red open squares),-L400C^DDM (green filled triangle), and -L400C^OG (green open triangle). Data of independent experiments (n ≥ 2) were subjected to 1-site-binding global fitting.

Fig. 2.

n-Octyl-β-d-glucopyranoside impairs Leu binding to the S2 site. (A and B) After 16-h incubation with 100 nM ³H-Leu, WT^DDM and WT^OG (A) and L400C^DDM and L400C^OG (B) were diluted in 50 mM NaCl-containing buffer (+Na), followed by the addition of ³H-Leu at different concentrations (10 nM, purple inverted open triangle; 25 nM, black filled circle; 50 nM, blue filled inverted triangle; 250 nM, red open square.) Data are from representative experiments performed in parallel. Error bars of triplicate determination were removed for clarity (SD ≤10%). (C) The S2 site binds Leu with high affinity. ³H-Leu was removed from the S2 site of LeuT-WT^DDM, resulting in ≈50% total binding, i.e., trapping of Leu in the S1 site (17) and increasing concentrations of ³H-Leu were added as shown in A. ³H-Leu equilibrium binding was normalized with regard to the initial binding (100%, i.e., before the dilution) and plotted as a function of [³H-Leu]. Data of 3 independent experiments were subjected to 1-site-binding global fitting, yielding a K_D^Leu for the S2 site of 23.1 ± 5.8 nM.

Fig. 3.

n-Octyl-β-d-glucopyranoside and TCA act as symport uncouplers. (A and B) LeuT-WT^DDM and WT^OG (A) and L400C^DDM and L400C^OG (B) were incubated with 100 nM ³H-Leu before dilution of the samples in 50 mM NaCl-containing buffer (+Na) followed by dilution into Na⁺-free buffer (−Na). Leu (symbols for Leu concentrations up to 250 nM are consistent with those in Fig. 2; 250 μM, green diamonds; 2.5 mM, magenta circles; 100 mM, black inverted triangles) or 500 μM clomipramine (CMI, blue open triangle) was added as indicated. Data are from representative experiments performed in parallel, and the error bars of triplicate determination were removed for clarity (SD ≤10%). (C) The half-time (t_1/2) of the release of ³H-Leu trapped in the S1 site decreases with increasing [Leu] binding to the S2 site. The t_1/2 from 2 independent experiments as depicted in A were plotted as a function of [Leu] and 3-parameter hyperbolic decay fitting of the data revealed an EC₅₀^Leu of 15.8 ± 2.7 nM. (D) Equilibrium binding of 100 nM ³H-Leu to LeuT-WT^DDM is inhibited by OG. Increasing concentrations of OG were added to a binding assay performed in DDM. ³H-Leu binding was decreased with increasing concentrations of OG and plateaued at 42.4 ± 1.8% with an IC₅₀^OG of 6.7 ± 0.8 mM. Data of 3 independent experiments were averaged and subjected to hyperbolic decay curve fitting and constants are shown ±SEM.

Fig. 4.

Identification of n-octyl-β-d-glucopyranoside in the S2 site of LeuT-E290S. (A) Overall distribution of OG in the P2₁ LeuT-E290S structure. The P2₁ structure of LeuT-E290S contains 2 molecules in the asymmetric unit (green and yellow cartoon representation). All OG molecules identified in the structure are highlighted with purple and red spheres (carbon and oxygen, respectively) and the location of leucine at the S1 site is shown with blue and red spheres (and magenta spheres for Na⁺). OG molecules bound at the S2 site are labeled with blue asterisks. (B) Schematic representation of a monomer of the LeuT-E290S structure is shown in yellow with the identified OG at the S2 site and the leucine-bound S1 site represented by spheres. (C) The unbiased F_obs − F_calc difference map calculated at 30- to 2.8-Å resolution for the P2₁ form of the LeuT-E290S structure before any modeling of the OG ligand at the S2 site. Other than the most exposed part of the glycoside head group, the OG model provides a snug fit to the residual density at the S2 site. (D) Electron density map of the S2 site in the LeuT-E290S structure. The final σ_A-weighted 2F_obs-F_calc electron density map displayed at 1σ contour level around the final model. The LeuT model is represented by stick representation with carbon atoms in yellow, nitrogen in blue, and oxygen in red. The OG model is displayed in a color-ramp according to the B-factor range 40–160 Ų (green to red) highlighting a poor order of the glycoside head group.

Fig. 5.

The conformational impact of ligand binding in the S2 site is different for substrates and inhibitors. (A) The conformational space explored during MD simulations by MD-WT-S1:Leu/S2:OG (purple-blue for protein backbone, with OG in magenta) and MD-WT-S1:Leu/S2:U (green) differs most at the extracellular portion of TM6 (TM6e). For comparison, the traces from these simulations are superimposed on the crystal structures WT-S1:Leu/S2:OG (cyan; PDB ID code 2A65) and WT-S1:Leu/S2:CMI (pink; PDB ID code 2Q6H). The superposition is based on the Cα atoms of TM3 and TM8, which differ least among these structures. (B) Superposition of MD-E290S-S1:Leu/S2:U (orange), MD-WT-S1:Leu/S2:U (green), and the crystal structure of E290S-S1:Leu/S2:OG (magenta), viewed from the same angle and using the same superposition scheme as in A. (C) Superposition of WT-LeuT structures. Note that with reference to TM6e of MD-WT-S1:Leu/S2:U, the TM6e of MD-WT-S1:Ala/S2:Ala (yellow), which has a substrate in the S2 site, moves in a direction opposite to that seen for this TM portion in the crystal structure WT-S1:Leu/S2:OG (cyan; PDB ID code 2A65). The simulation results are shown as traces in A and as average structures in B and C. (D) Top view of the superposition of MD-WT-S1:Ala/S2:Ala and the crystal structure WT-S1:Leu/S2:OG, WT-S1:Leu/S2:CMI, and E290S-S1:Leu/S2:OG, with the same color coding as defined in A–C. (E) Definition of the inter-TM angles, illustrated for A_6e-10e. The cylinders represent the identified TM segments in the exact configurations and color coding shown in D. Note that the reference position (cyan) is defined by TM3e and TM10e, and the position of TM6e is shown for each of the compared structures. The usual definition of the angle between noncoplanar segments is illustrated for the WT-S1:Leu/S2:OG (cyan) structure. Line a is the perpendicular connecting TM6e and TM10e, which are noncoplanar; line b is the projection of TM6e on the plane that is perpendicular to a and intersects with TM10e.