Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Abstract

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

Keywords: LeuT; aquifex aeolicus; biophysics; neuroscience; neurotransmitter transporters; solid state NMR; structural biology; substrate binding site.

Figure 1.. Assessment of L-leucine binding to LeuT WT by solid state NMR.

(A) Cartoon illustration of experimental approach. ¹⁵N enriched L-leucine substrate is added to detergent reconstituted LeuT, which is subsequently crystallized using large scale sitting drop vapour diffusion. Rod-shaped microcrystals form within 24 hr and can be readily harvested. (PDB ID: 3F3E) (B) LeuT WT purified in NaCl (red) and LeuT purified in KCl (black). ¹⁵N L-Leucine specific peak is indicated by an asterix with a chemical shift of 38.2 ppm. Spectra are tentatively intensity normalized to the ¹⁵N natural abundance signal from the LeuT backbone amides. Signal-to-noise is calculated to be 21. (C) ²³Na-NMR of LeuT WT (red) and LeuT WT in KCl (black) in presence of leucine. Minor peak at −8.9 ppm represents the shape of one or two structural sodium molecules. Despite inequivalent location of the two sodium sites in the LeuT, the coordination mechanism is almost identical which might account for the observation of a single peak in the ²³Na-NMR spectrum instead of two distinct peaks. DOI: http://dx.doi.org/10.7554/eLife.19314.003

Figure 1—figure supplement 1.. Functional characterization of LeuT WT.

LeuT WT was purified in KCl and eluted in 0.05% DDM (pH 8.0). Scintillation proximity assay-based measurements of (A) [³H]leucine saturation binding to 100 ng LeuT in the presence of 200 mM NaCl and (B) Na⁺-dependent [³H]leucine binding (100 nM) by 100 ng LeuT. Ionic strength was compensated with KCl. Data are displayed as means ± s.e.m., performed in triplicates, n = 3. DOI: http://dx.doi.org/10.7554/eLife.19314.004

Figure 1—figure supplement 2.. Microscopy image of LeuT microcrystals.

LeuT microcrystals are needle-shaped and have a length of 1–10 μm. The microscopic image was solely used to assess the quality of the microcrystalline material. DOI: http://dx.doi.org/10.7554/eLife.19314.005

Figure 1—figure supplement 3.. 1D 15N CP/MAS spectrum of frozen and lyophilized LeuT WT samples.

(A) Protein concentration: 2 mg/ml, leucine concentration: 1 mM. The spectrum is dominated by the signal arising from the free unbound leucine, at ~42 ppm. Spectrum was recorded on a 400 MHz Bruker shielded wide bore magnet equipped with a 3.2 mm MAS HCN operating at ~100 K. Spinning rate: 8000 kHz. CP contact time: 1000 us, recycling delay: 3 s. 24 k scans were required to record presented spectrum. (B) ¹⁵N CP/MAS spectra from dry L-leucine powder (top panel) and from lyophilized leuT samples (bottom panel). Free ¹⁵N L-Leucine has a distinct chemical shift at 118 ppm. For the LeuT WT NaCl preparation (red) several additional peaks appear around the dominating free state peak. Though these peaks vary slightly in intensity when compared to LeuT WT KCl (dark). These peaks do not originate from structural leucine. Spectra were recorded on a 700 MHz Bruker shielded wide bore magnet equipped with a 4 mm MAS HCN probe operating at 298 K. Spinning rate: 12500 KHz, CP contact time: 1500 us, recycling delay: 2.5 s. 65 K scans were required to record presented spectra. DOI: http://dx.doi.org/10.7554/eLife.19314.006

Figure 1—figure supplement 4.. 15N L-leucine spectrum substrate peak for LeuT WT.

(A) Line broadening for window function 1 Hz, Signal-to-noise in calculated to be 21, Full width half height (FWHH) of the substrate peak is 31 Hz. (B) Line broadening for window function 10 Hz. DOI: http://dx.doi.org/10.7554/eLife.19314.007

Figure 1—figure supplement 5.. In-solution 1D ¹⁵N spectra of free 98% ¹⁵N L-leucine at different pH.

(A) pH titration of the L-leucine amine. The 1D ¹⁵N spectra are recorded for 1024 scans at 25°C. (B) ¹⁵N chemical shift of the L-leucine amine as a function of pH. From the sigmoidal curve fit the pI is estimated to be 9.72 ± 0.06. DOI: http://dx.doi.org/10.7554/eLife.19314.008

Figure 1—figure supplement 6.. 1D 13C CP/MAS spectrum of microcrystalline LeuT WT samples.

Spectra for samples prepared in NaCl or KCl are colored in red and black, respectively. Resonances originating from labelled leucine are indicated by their chemical shifts and their respective assignments. These results are in good agreement with the ¹⁵N 1D spectra, and only one set of chemical shifts from the ligand can be observed. DOI: http://dx.doi.org/10.7554/eLife.19314.009

Figure 2.. Effects of S1 and S2 site mutations on the L-leucine chemical shift .

(A–B) Cartoon representation displaying the location of F253 in the S1 site and L400 in the proposed S2 site based on PDB: 3USG (Wang et al., 2012a). (C) ¹⁵N 1D NMR spectrum of LeuT WT (red), F253A (green) and L400S (blue). Inset: Close-up of L-leucine specific peak. Spectra are tentatively intensity normalized to the ¹⁵N natural abundance signal from the LeuT amides. DOI: http://dx.doi.org/10.7554/eLife.19314.010

Figure 2—figure supplement 1.. Comparison of spectra derived from LeuT WT purified and crystallized in either 1 mM (red) or 5 uM (purple) free leucine.

Spectra are tentatively normalized to the LeuT natural abundance amide signal. As expected the substrate peak relates to LeuT concentration and not to the added free leucine concentration. DOI: http://dx.doi.org/10.7554/eLife.19314.011

Figure 2—figure supplement 2.. Power spectra of LeuT WT (red), F253A (green) and L400S (blue).

To rule out that wrong phasing would cause the chemical shift difference observed for the substrate peak of the spectra presented in Figure 2C, we present the them also as power spectra (which is ultimately a squared magnitude spectra) where phase signs are not preserved. We have intensified the F253A signal to only demonstrate the chemical shift difference. DOI: http://dx.doi.org/10.7554/eLife.19314.012

Figure 2—figure supplement 3.. Comparison of LeuT WT and LeuT F253A in the presence of 1 mM free substrate.

(A) Full spectrum displayed with a line broadening of the windows function of 100 Hz. Tentatively intensity normalized using natural abundance signals. (B and C) Close up of the substrate peak region showing preserved chemical shift difference between the substrate bound in LeuT WT (black dashed line) and LeuT F253A (blue dashed line) in two different free substrate concentrations. DOI: http://dx.doi.org/10.7554/eLife.19314.013

Figure 2—figure supplement 4.. Cartoon representation of S1 bound substrate (green).

Sodium ions are depicted in blue. Measured chemical shift values in red (in ppm) and measured distances in black (in Å). Image is made from PDB file: 3F3E. DOI: http://dx.doi.org/10.7554/eLife.19314.014