A Tour of LeuT


Please be patient while the structures in the left frame load. In order to display all of the structures in the tour properly, press 'View' buttons below in order (from 1 to the end).


The leucine transporter (LeuT) belongs to a class of transmembrane proteins that use the electrochemical potential of specific ions to catalyze the transport of various organic substrates across the cell membrane. In eukaryotic organisms, these proteins play a very important role in neural signalling and their malfunction is believed to be a cause of many neural diseases.

This tour explores the structure and function of a bacterial leucine transporter isolated from Aquifex aeolicus (LeuTAa).[1] LeuTAa utilizes the existing electrochemical potential of Na+ ions to facilitate the uptake of amino acid leucine. The intracellular concentration of Na+ is lower than the extracellular concentration of Na+. The concentration difference creates an electrochemical potential gradient that is used to catalyze the uptake of organic substrates (in this case leucine). The transport is not dependent on any other source of energy (for example, ATP). The LeuTAa reaction is:

2Na+out + leucineout → 2Na+in + leucinein [2]

The human homologue of LeuTAa is dependent on Cl- as well as Na+ concentration: they belong to a class of Na+/Cl- dependent transporters from the solute carrier 6 (SLC6) family (also referred to as neurotransmitter sodium symporters, or NSS). They transport many biologically important monoamines, for example dopamine (DA), noradrenaline and serotonin. The human SLC6 transporters are also primary targets for addictive substances (cocaine and amphetamine) as well as for drugs for the treatment of central nervous system disorders [3].

The LeuTAa transporter consists of two identical subunits (it is a homodimer), one subunit is coloured yellow, the other blue. (Unless otherwise noted, in each view the extracellular side of the transporter structure is always pointing upwards.)

This is a look at the homodimer from the extracellular space. The two subunits are approximately related by a two-fold symmetry axis located between two subunits, perpendicularly to this screen.

The transporter is built up of predominantly non-polar (hydrophobic) residues (white areas on the structure). This hydrophobic character of the protein surface complements well the hydrophobic character of the cell membrane. To see the electrostatic surface potential of LeuTAa click on image thumbnail below.

Since the two subunits are identical, the remainder of the tour will concentrate only on one subunit.

The secondary structure of LeuTAa is composed of twelve transmembrane (TM) alpha helix segments (green and red). There are two very short β-sheets (blue) and numerous loops (yellow). The two red α helices, TM9 and TM12, are points of contact between the two subunits in the homodimer. This is a unique fold among membrane proteins but it is typical for the SLC6 family of Na+-dependent symporters. The monomer is about 70 Å tall and approximately 48 Å in diameter.

When bound inside the protein, the substrate (leucine, in sticks) and two sodium cations (purple spheres) are located approximately in the middle of the cell membrane. Although it appears that all three are buried deep inside the protein structure, they are actually very close to the outer protein surface. This is because the protein's structure resembles a bowl approximately 6 Å deep with its open side facing the extracellular space with its bottom close to the mid-way between the extra- and intracellular space. Click on the thumbnail below to better visualize the overall monomer shape and the location of leu and Na+ ions inside the protein.

The residues located on the transmembrane segments (TM) 1, 3, 6 and 8 are involved in binding leucine and sodium ions.

There are two different sodium sites, Na1 and Na2. The Na1 site contains a six coordinate Na+ ion. All donor atoms are oxygens. Five donor atoms are provided by the residues located on TM1 (Ala22 and Asn27) and TM6 (two oxygens from Thr254 and one from Asn286). The sixth coordination site is occupied by the oxygen atom from deprotonated carboxyl group on leucine.

This primary coordination sphere results in a distorted octahedral geometry at the Na1 site.

The Na2 site contains a five coordinate Na+ ion. Again, all donor atoms are oxygen atoms. Five different residues provide the donor atoms: Glu20, Val23 (both on TM1), Ala351, Thr354 and Ser355 (all on TM8).

This primary coordination sphere produces a distorted trigonal bipyramidal geometry at Na2 site. Click on the tumbnail below to see the Na+-O distances for both Na sites.

The mechanism behind the selectivity for Na+ over K+ found in these proteins is not fully understood. For the Na1 site, the presence of negatively charged ligand (Leu) is considered important for selectivity because it can off-set relatively high dehydration energy for Na+ cation and would prefer a cation with higher charge-to-radius ratio. The 'snug-fit' provided by a chelating Thr254 further increases the preference for the smaller sodium cation. The low coordination number of the Na2 site and ligands located close in the protein primary structure (forming a small-size cavity) are two likely features that dictate the preference of this site for Na+ [4].

The Leu binding pocket is designed to stabilize both polar (carboxylic and NH2 groups) and non-polar (isopropyl group) ends of the molecule. The polar end is stabilized through polar interactions (yellow lines indicate selected polar leucine-protein interactions). The most noteworthy interaction is between the carboxylate on leucine and Na+ at the Na1 site (left hand side of the display).

The non-polar isopropyl group is stabilized inside large hydrophobic pocket formed by two phenyl groups from Phe253 and Phe259, isopropyl group from Val104 and hydrocarbon chain on Ile359.

The LeuTAa transporter has two gates: one opens to the extracellular space the other to the intracellular space. Two pairs of amino acids, a pair for each gate, serve as the 'gate keepers': red pair (top) for the extracellular and blue pair (bottom) for the intracellular gate. In this structure, both gates are closed and it is said that the LeuTAa is in occluded-state.

Arg30 and Asp404 are the extracellular gate keeper pair. They keep the gate locked through H-bonding with two water molecules. This pair can be found at the bottom of the bowl-shaped structure (click on the tumbnail below to see the location of this gate).

The intracellular gate keeper pair is Arg5 and Asp369. The two amino acids close the gate also through hydrogen bonding. However no water molecules are involved in the functioning of this gate.

The transporter's extracellular gate can be locked in open state (open-to-out protein conformation) if leucine is replaced with the LeuTAa inhibitors. The display on the right shows the LeuTAa structure with one possible inhibitor - tryptophan (ball-and-stick model in the middle).

Tryptophan's indole ring is considerably larger (bulkier) than leucine's isopropyl group. Consequently, there is a mismatch in size between the Trp ring and the hydrophobic pocked of the substrate binding site and the extracellular gate residues are pushed away preventing the formation of H-bonds required to lock the gate. Click on the thumbnail below to see the difference between occluded-state and open-to-out tertiary structures.

The open-to-out structure reveals the presence of another tryptophan molecule located about 4 Å above the first one. This binding site likely represents a low-affinity site for the transporter's substrate, a site that is transiently occupied as the substrate molecule enters its binding pocket. The second Trp molecule forms hydrogen bonds with the amino acids of the outer gate. It is probable that this hydrogen bonding serves to direct and dehydrate substrate as it enters the transporter.

This is the structure of Trp- bound LeuTAa. Feel free to explore it further.



The structures of Leu and Trp bound LeuTAa were reported by Singh, S.K. et al. in Science 2008, 322, 1655 (PDB IDs 3F3E - Leu bound LeuTAa and 3F3A - Trp bound LeuTAa).

Copyright: Robert Morris and Alen Hadzovic, 2011.



[1] Aquifex aeolicus is is one of the most thermophilic bacteria known: it grows at 95oC in hot water springs. It completely relies on inorganic materials as both energy and carbon sources. For more information, see the A. aeolicus entry in the Encyclopedia of Life.

[2]The sodium : substrate stoichiometry varies depending on transporter: norephedrine and serotonin transporters have 1 : 1 sodium-to-substrate ratio, while glycine transporter requires 3 : 1. However, recently these values have been questioned (see discussion in Noskov, S.Y. and Roux, B. 2008 377, 804-818).

[3]For more details see Wang, C.-I., & Lewis, R.J. Biochemical Pharmacology 2010 79, 1083-1091.

[4]For more details on cation selectivity see Noskov, S.Y. & Roux, B. 2008 377, 804-818 and Yu, H.; Noskov, S.Y. & Roux, B. Proc. Nat. Ac. Sci. 2010 107, 20329-20334.