Research ArticleBIOPHYSICS

Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT

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Science Advances  11 May 2018:
Vol. 4, no. 5, eaar6179
DOI: 10.1126/sciadv.aar6179
  • Fig. 1 Measuring the HDX in distinct LeuT regions by mass spectrometry.

    (A) Online pepsin proteolysis yielded a total of 67 LeuT peptides suitable for local HDX-MS analysis. The identified peptides are depicted as black bars and are aligned with the corresponding LeuT sequence. The peptides cover 71% of the protein sequence. Individual structural motifs in LeuT are indicated above the protein sequence in gray color. (B) Representative mass spectra for individual LeuT peptides that cover the N terminus (peptide 1–12), the substrate binding site in TM 6 (peptide 253–260), and EL4 (peptide 315–325). Mass spectra at the 0-min time point derive from unlabeled samples. The isotopic envelopes then shift to higher values on the m/z (mass-to-charge ratio) scale as a function of time (0.25 to 60 min) and ion/substrate composition (that is, K+, Na+, and Leu state) due to deuterium incorporation at the backbone amide position. Binding of Na+ or the combination of Na+ and leucine led to decreased HDX (structural stabilization) in the substrate binding site in TM 6 (peptide 253–260, blue panel) and to increased HDX (structural destabilization) in EL4 (peptide 315–325, red panel) relative to the K+ state. The exchange rate in the N terminus (peptide 1–12, gray panel) was not affected upon binding of Na+ and leucine.

  • Fig. 2 Pinpointing LeuT segments involved in substrate transport.

    The average relative deuterium uptake of the Na+ state (A) or the Leu state (B) is subtracted from the average value of the K+ state for each peptide and time point. Individual LeuT peptides are plotted on the y axis in an ordered manner starting from the N to the C terminus. Differences in HDX (ΔHDX, colored lines) between the Na+- or Leu-bound state and the K+ state are plotted on the x axis. Positive and negative values indicate increased and decreased HDX in the K+ state, respectively. Values represent means of three independent measurements. Gray bars illustrate the sum of ΔHDX values for all sampled time points. Structural motifs in LeuT, for which we observed Na+- and/or leucine-induced changes in HDX, are annotated along the y axis. (C) Differences in HDX between the Leu state and the K+ state in (B) are mapped onto the LeuT crystal structure (pdb 2A65) displaying a substrate-bound, outward-facing occluded conformation. Red and blue colored regions indicate LeuT segments that became more dynamic (increased HDX) or exhibited structural stabilization (decreased HDX) in the Leu state, respectively. LeuT segments that were not affected upon Na+/substrate binding or for which no HDX data could be obtained are colored in gray. The same color scheme is applied to the topology map of LeuT in (D).

  • Fig. 3 Partial unwinding of individual helices in LeuT.

    (A) LeuT segments that exchanged via an EX1 regime or that exhibited signs thereof are colored in orange and yellow in the LeuT crystal structure (pdb 2A65), respectively. The same color scheme is applied to the topology map of LeuT in (B). (C) Representative mass spectra for peptide 184–199, which covers the intracellular half of TM 5, for the K+, Na+, and Leu states and all sampled time points. The bimodal isotopic envelopes can be fitted to a low-mass (blue) and high-mass (red) population. Addition of Na+ or the combination of Na+ and leucine substantially decreased the rate of correlated exchange, that is, the conversion of the low-mass population to the high-mass population, relative to the K+ state. Na+- and leucine-induced stabilization of transmembrane helices was also evident for TMs 1a/6/7, with structural stabilization being consistently more pronounced in the Leu state than in the Na+ state (fig. S7).

  • Fig. 4 Extraction of kinetic measures for individual unfolding events in LeuT.

    (A) Time-resolved HDX data (K+ state) for different LeuT peptides covering EL4, TMs 1a/5/7, and the substrate binding site in TM 6. The bimodal isotopic envelopes can be fitted to a low-mass (colored) and high-mass (gray) population. (B) The average relative abundance of the low-mass population is plotted against labeling time for each peptide in (A). Values represent means ± SD of three independent measurements. The data points for each peptide are fitted to an exponential decay function (see the Supplementary Materials). Note that each peptide is color-coded as specified in (A). (C) Tabular overview of the calculated kinetic parameters (that is, kop and the half-life of the low-mass population) for each LeuT peptide. The best-fit values are reported together with the values defining the 95% confidence interval (in square brackets). Notably, the calculated kop values for individual unfolding events in LeuT differed by as much as two orders of magnitude.

  • Fig. 5 Impact of K+ on the conformational ensemble of LeuT.

    (A) The average relative deuterium uptake of the K+ state is subtracted from the average value of the Cs+ state for each peptide and time point. Individual peptides are plotted on the y axis in an ordered manner starting from the N to the C terminus. Differences in HDX (ΔHDX, colored lines) between the two states are plotted on the x axis. Positive and negative values indicate increased and decreased HDX in the Cs+ state, respectively. Values represent means of three independent measurements. Gray bars illustrate the sum of ΔHDX values for all sampled time points. Structural motifs in LeuT, for which we observed K+-induced changes in HDX, are annotated along the y axis. (B) Same representation as in (A) for the comparison of the K+ and K+High states. Positive and negative values indicate increased and decreased HDX in the K+ state, respectively. Values represent means of three independent measurements. Notably, (A) and (B) are consistent with a concentration-dependent, K+-induced closure of LeuT to the extracellular environment.

  • Fig. 6 Proposed substrate transport mechanism in LeuT.

    (1) Under apo state conditions, LeuT preferentially assumes an outward-facing open conformation with relatively high Gibbs free energy. (2) HDX is decreased upon Na+ binding, suggesting stabilization of the protein backbone in basically the same transporter conformation as for apo state, presumably the outward-facing open. (3) According to crystal structures (5), binding of a transportable hydrophobic amino acid (for example, leucine) prompts occlusion of the extracellular vestibule. EL4 and the water-mediated salt bridge between Arg30 and Asp404 prevent water access to the substrate binding site. On the basis of HDX data, it appears that the substrate-bound, outward-facing occluded state represents the thermodynamically most favorable LeuT conformation in the transport cycle. (4) As shown for MhsT (11) and supported by the results herein, the concurrent unwinding of the intracellular halves of TMs 1 and 5 creates a solvent pathway for intracellular release of the Na2 ion, which triggers transition of LeuT to an inward-facing occluded transporter conformation (5). (6) The rates for the observed EX1 kinetics suggest that the release of both the substrate molecule and the Na1 ion is facilitated by a partial unwinding of the intracellular part of TM 7, thereby enabling subsequent isomerization of LeuT to an inward-facing open state (7). Notably, binding of intracellular K+ to LeuT (not indicated in the diagram) potentially prevents substrate rebinding as well as isomerization of LeuT from an inward-facing open (7) to an outward-facing open conformation (1) through a poorly understood return mechanism.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaar6179/DC1

    Supplementary Materials and Methods

    fig. S1. Na+-dependent binding of [3H]leucine to purified LeuT.

    fig. S2. Correlation between the measured HDX and secondary structure elements in LeuT.

    fig. S3. Deuterium uptake plots for detergent-solubilized LeuT.

    fig. S4. Impact of different leucine concentrations on local HDX rates in LeuT.

    fig. S5. Partial unwinding of individual helices in LeuT reconstituted into phospholipid-containing bilayer nanodiscs.

    fig. S6. Sequence coverage map for LeuT reconstituted into phospholipid-containing bilayer nanodiscs.

    fig. S7. Na+- and substrate-induced stabilization of TM helices.

    fig. S8. Compared conformations of EX1 segments in crystal structures.

    table S1. RMSD and hydrogen bond variations between three x-ray structures of LeuT for the EX1 segments.

    table S2. Detailed hydrogen bond analysis in three x-ray structures of LeuT for backbone amide groups in the EX1 segments.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Na+-dependent binding of 3Hleucine to purified LeuT.
    • fig. S2. Correlation between the measured HDX and secondary structure elements in LeuT.
    • fig. S3. Deuterium uptake plots for detergent-solubilized LeuT.
    • fig. S4. Impact of different leucine concentrations on local HDX rates in LeuT.
    • fig. S5. Partial unwinding of individual helices in LeuT reconstituted into phospholipid-containing bilayer nanodiscs.
    • fig. S6. Sequence coverage map for LeuT reconstituted into phospholipid-containing bilayer nanodiscs.
    • fig. S7. Na+- and substrate-induced stabilization of TM helices.
    • fig. S8. Compared conformations of EX1 segments in crystal structures.
    • table S1. RMSD and hydrogen bond variations between three x-ray structures of LeuT for the EX1 segments.
    • table S2. Detailed hydrogen bond analysis in three x-ray structures of LeuT for backbone amide groups in the EX1 segments.

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