Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 May;21(5):472-9.
doi: 10.1038/nsmb.2816. Epub 2014 Apr 20.

Conformational dynamics of ligand-dependent alternating access in LeuT

Affiliations

Conformational dynamics of ligand-dependent alternating access in LeuT

Kelli Kazmier et al. Nat Struct Mol Biol. 2014 May.

Abstract

The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na(+)- and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Model of LeuT alternating access inferred from the crystal structures
(a) Root mean square deviation (RMSD) between occluded (PDB ID: 2A65) and outward-open (PDB ID: 3TT1) LeuT crystal structures mapped onto the outward-facing substrate-occluded structure. The inset is a close-up view to highlight regions of most substantial differences, TMs 1, 6, and EL4, as well as TM7. (b) RMSD between occluded (PDB ID: 2A65) and inward-open (PDB ID: 3TT3) LeuT crystal structures mapped onto a ribbon diagram of the outward-facing substrate-occluded structure. The regions of most appreciable differences (TMs 1 and 5) as well as TMs 6 and 7 are shown in a close up view.
Figure 2
Figure 2. Na+-induced opening and Na+- and Leu-induced closing of the LeuT extracellular side
(ad) Distance distributions, depicting the probability of a distance P(r) versus distance (r) between spin labels, and reporting the conformational dynamics of EL4, TM6, TM1, and TM7 on the extracellular side of LeuT. The locations of representative spin label pairs are highlighted on the substrate-occluded structure by black spheres connected by a line. TM helices expected to respond to ligand binding are shown as colored cylinders. The approximate location of the extracellular vestibule is colored cyan. Distance distributions for each pair were obtained in the apo, Na+-bound (Na+), and Na+- and Leu-bound (Na+ and Leu) intermediates. The multi-component distributions reflect multiple conformations of LeuT in equilibrium. For illustration, we simulated the distance components corresponding to the outward-open (O) and outward-closed (C) conformations using the average distance and width of each component. The resulting Gaussians are superimposed in gray. The shift in the conformational equilibrium of EL4, TM6a, TM1b and TM7b relative to static reference points are shown in a, b, c, and d respectively.
Figure 3
Figure 3. Fluctuation dynamics of TMs 6, 7 and the N-terminal segment mediate the opening of the intracellular side of LeuT
(ad) Distance distributions, depicting the probability of a distance P(r) versus distance (r) between spin labels, and reporting the conformational dynamics of IL3, TM7, TM1, and TM5 on the intracellular side of LeuT. Distance distributions for each pair were obtained under three conditions as in Figure. 2. Here, the simulated grey distributions reflect inward-open (O) and inward-closed (C) conformations. (a,b) TM7 and IL3 distributions indicate an equilibrium between two conformations that is modulated by Na+ and substrate binding. (c,d) In contrast, distributions for TMs 1 and 5 do not indicate ligand-dependent conformational changes.
Figure 4
Figure 4. β-OG stabilizes the outward-facing conformation of LeuT in the presence of Na+ and leucine
(a) Close-up view of LeuT extracellular vestibule showing the simultaneous binding of leucine (red), Na+ (blue) and β-OG (yellow) (PDB ID: 3GJD). Comparison of distance distributions in β-DDM (b) and β-OG (c) demonstrate that the latter stabilizes an outward-facing conformation on the extracellular side and a closed conformation on the intracellular side (TM1a and 3). The corresponding distance component is indicated by an arrow. The component labeled * arises from aggregated protein during the concentration process. (d) Predicted distance distributions from three LeuT crystal structures (3TT1: outward-facing, 2A65: substrate-occluded, 3TT3: inward-facing) using MMM (see methods).
Figure 5
Figure 5. The Y268A or R5A mutations induce structural rearrangements in LeuT
(a) Close up view of the putative intracellular gate showing the network of charge interactions stabilized by Tyr268 and involving Arg5. TMs 1 and 5, are highlighted. (b,c) the mutations Y268A and R5A lead to the appearance of new distance components in the distributions of TMs 1 and 5 (dashed lines, c) that is not present in the WT background (b) under apo conditions.
Figure 6
Figure 6. Models of LeuT conformational changes derived from restrained ensemble simulations
(ac) Extracellular view of the occluded (red) and outward-facing (blue) models of LeuT, derived from the DEER restraints, superimposed on (b) the substrate-occluded crystal structure (2A65, orange) and (c) the inward-facing crystal structure (3TT3, purple). Prominent displacements of TMs 1 and 6 (red model) to close the extracellular vestibule are observed in the occluded conformation relative to the crystal structures. (df) Intracellular view of the inward-facing model (black), derived from the DEER restraints, superimposed on (d) the outward-facing model (blue), (e) the substrate-occluded structure (2A65, orange) and (f) the inward-facing structure (3TT3, purple).
Figure 7
Figure 7. Cartoon model of LeuT transport derived from EPR data
Transmembrane helices involved in conformational changes at each step in the transport cycle are highlighted in color. (a) The cycle begins following release of ion and substrate to the intracellular side (In). Apo-LeuT samples inward- (a) and outward-facing conformations (b). (b) Na+ binding favors opening of the extracellular side (Ex) through shifts in the equilibrium of the extracellular motifs. Coupled closing of the intracellular side involves a shift in the equilibrium of the intracellular motif to its closed position, which stabilizes the intracellular gate. (c) Leu binding at the S1 site, and presumably at the S2 site as well, causes a large scale closure of the extracellular side leading to an occluded state. (d) Fluctuations on the intracellular side, facilitated by the unwound region of TM6 and a kink at Gly294 of TM7 mediate the opening of the intracellular side. (e) Na+ dissociates to the intracellular solution where its concentration is low. (f) In the absence of bound Na+, leucine affinity to LeuT is reduced driving its dissociation to the intracellular side. The cycle continues through the isomerization from inward-facing (f) to outward-facing (a).

References

    1. Rudnick G. Mechanisms of Biogenic Amine Neurotransmitter Transporters. In: Reith MA, editor. Neurotransmitter Transporters. Humana Press; 2002. pp. 25–52.
    1. Iversen L. Neurotransmitter transporters and their impact on the development of psychopharmacology. Br J Pharmacol. 2006;147(Suppl 1):S82–S88. - PMC - PubMed
    1. Amara SG, Sonders MS. Neurotransmitter transporters as molecular targets for addictive drugs. Drug Alcohol Depend. 1998;51:87–96. - PubMed
    1. Singh SK. LeuT: A prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure. Channels (Austin) 2008;2 - PubMed
    1. Gether U, Andersen PH, Larsson OM, Schousboe A. Neurotransmitter transporters: molecular function of important drug targets. Trends Pharmacol Sci. 2006;27:375–383. - PubMed

Methods References

    1. Quick M, Javitch JA. Monitoring the function of membrane transport proteins in detergent-solubilized form. Proc Natl Acad Sci U S A. 2007;104:3603–8. - PMC - PubMed
    1. Pannier M, Veit S, Godt A, Jeschke G, Spiess HW. Dead-time free measurement of dipole-dipole interactions between electron spins. Journal of Magnetic Resonance. 2000;142:331–40. - PubMed
    1. Jeschke G. DEER distance measurements on proteins. Annu Rev Phys Chem. 2012;63:419–46. - PubMed
    1. Jeschke G, et al. DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data. Applied Magnetic Resonance. 2006;30:473–498.
    1. Chiang YW, Borbat PP, Freed JH. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. Journal of Magnetic Resonance. 2005;172:279–95. - PubMed

Publication types