LeuT
Created by Kendrick Smith
Elucidating the mechanism of small molecule transport across membranes continues to pique the interest of researchers. Reuptake, the process of reabsorbing neurotransmitters after use in neural synapse, relies on the fundamental process of small molecule transport (3). The leucine transporter (LeuT,3f3a), isolated from prokaryotic Aquifex aeolicus, serves as a convenient research model of the reuptake process. LeuT utilizes potential energy stored in electrochemical gradients in order to couple transport of small hydrophobic amino acids, such as leucine, glycine, and alanine (1). This gradient, consisting of a net positive charge in the extracellular space resulting from sodium ions and the net negative charge in the intracellular space, results in the net flux of sodium ions into the cell. LeuT permits two sodium ions and one substrate amino acid into the active site which structurally resembles a canal. Structural analogies between LeuT and similar reuptake symporters found in humans encouraged structural analysis of LeuT in complex with various tricyclic antidepressants (TCAs).
The molecular weight of LeuT is 58031.91 Da and has an isoelectric point of 8.96 as determined from Expasy, which calculated the theoretical weight and pI based off the amino acid sequence by looking at residue weight. LeuT itself is homodimeric, or consists of two identical subunits. Each monomer is 70 Angstroms long and roughly 48 Angstroms wide. Furthermore, each subunit contains a substrate tunnel leading down to the active site (4). Modification of the tunnel space allows translocation of the substrate. There are three main conformations of the subunits: “open-to-out”, occluded state, and the “open-to-in” state (1). The open-to-out state exposes the entrance of the substrate tunnel to the extracellular cytosol, allowing incorporation of substrate and sodium ion ligands. Due to the bowl-like shape of the protein the ligand/substrate sits relatively close to the outer surface of the protein. The occluded state is achieved when both an extracellular gate and an intracellular gate are closed and the substrates are locked inside of the protein. Finally, the open-to-in conformation releases the substrate and sodium ions into the intracellular periplasm. The inhibition of this process factors in at changing the open-to-out conformation. A theoretical competitive inhibitor would involve a competitive substrate molecule that would be able to enter the binding site of the protein however not allow for translocation by locking the protein in the occluded state (1). Non-competitive inhibitors have been found. Tryptophan serves as an example non-competitive inhibitor of the process. The bulky indole ring of the side chain purports a size mismatch which necessarily denies the amino acid access to the active site (1). Hydrogen bonds form between the indole nitrogen of the amino acid and the various residues that constitute the extracellular gate. This interaction locks LeuT in its open-to-out conformation (1).
The electrostatic surface potential directly reflects on the fact that LeuT is an integral membrane protein (4). Integral membrane proteins are located as part of a cellular membrane, and generally perform the same role as LeuT, active transport of small/large, polar/nonpolar molecules into and out of the cell or organelle. Another example of such protein would be ATP/ADP translocase which specifically couples ion gradients with pushing ATP out of intracellular matrices of mitochondria. Because it is an integral membrane protein, the distribution of critical surface residues affords the protein key electrostatic advantages. The extracellular portion of the protein carried a slight negative charge which attracts positively charged sodium cation whereas the intracellular surface had a partial positive charge to expel the ligand and substrate (4). The space between the two surfaces consists primarily of hydrophobic residues which complements the hydrophobic character of the middle portion of plasma membranes, containing the oily nonpolar residues of phospholipids. These surface potentials directly reflect on the character of the protein, an integral membrane protein (4).
The secondary structure of each subunit consists primarily of alpha helices. Each subunit contains twelve α-helices. There are four alpha helices, two per subunit, which interacts with the other vicinal subunit, thus holding the protein together. Four α-helices are responsible for the conformational change which expels the ligand and substrate out of the binding pocket. These helices themselves are kinked at a specific point which gives the appearance of a cup where the ligand and substrate nestle. Therefore, these four α-helices work very similar to a rocker, pivoting around the kink in the helical structure, and this pivoting is what differentiates between the open-to-out conformation of the LeuT/substrate complex and the open-to-in conformation of the LeuT/substrate complex, with the solvent accessible tunnel available in the intracellular space (3).
Residue functionality has a key role in the activity of LeuT, specifically with defining conformation and stabilizing the substrate in the substrate tunnel. As stated above, the occluded state of LeuT is defined from the closing of two cellular gates, the extracellular gate and the intracellular gate. The extracellular gate consists of two amino acids, arginine-30 and aspartic acid-404 (4). Incorporation of random water molecules through hydrogen bonding with the residues forms the gate. Furthermore, roughly 4 Angstroms above the active site, another tryptophan is present which can serve as an auxillary, transient binding site for substrates (1,4). This tryptophan interacts with the residues of the outer gate to allow dehydration and direct the substrate during its path into the substrate tunnel.The intracellular gate is made of R5 and D369, without the use of water molecules. This suggests that the two participate in hydrogen bonding with themselves to protect the substrate tunnel from adsorbing various molecules from the intramembrane cytosol, such as small molecule amino acids that were recently transferred.
The most notable interactions occur in the active site where hydrogen bonding, hydrophobic effects, and ionic effects dictate substrate stabilization. Helices 1,3,6 and 8 all contain residues specifically designed to mediate ligand/substrate incorporation. There are two binding sites for sodium. One of the binding sites is occupied with a sodium stabilized by electrostatic interactions between residues G20, V23 A351, T354 and S355, where the stabilization occurs through interactions with the carbonyl oxygen of the amide bond (2). However, the second sodium binding site holds more important due to an important interaction with the substrate hydrophobic, small molecule amino acid. The second sodium holds two key coordinated interactions with the carbonyl oxygen of T254 and the side chain hydroxyl group. Furthermore, it coordinates with the free carboxylate of the substrate amino acid. This coordination helps to stabilize the amino acid in the binding pocket, anchoring it electrostatically to the protein itself. As a result, a coordination shell develops around the sodium ion which size is most compatible with smaller charged metallic species, thus ruling out other typical synapse metal cations such as K+ from being a viable option (5).
Substrate binding functions similarly to the typical “lock-fit” mechanism: substrates that can fit into the binding pocket will best be suited for protein function. Aforementioned, the substrate is stabilized through ionic interactions between the free carboxylate of the substrate amino acid and one of the sodium ions present in the substrate tunnel (4). The non-polar side chains are nestled into a hydrophobic pocket composed of F253, F259, V104, I359 (1). The logic behind this design follows the standard “like-dissolves-like” motif found in general chemical academia. The size of the pocket does limit the type of substrate that can be incorporated. It has been shown that with increasing side chain size, the volume strain placed on the binding pocket can cause key electrostatic interactions and hydrogen bonds to be disrupted leading to severe conformational changes. Phenylalanine appeared to have been the largest of the non-polar amino acids that could be translocated via LeuT, however with decreased efficiency. Attempts at transport of tryptophan lead to a widening of the binding pocket, directly freezing LeuT in the open-to-out conformation. “Good” substrates included leucine, alanine, and methionine to an extent due to the sulfur induced dipole (1).
LeuT is a prokaryotic, bacterial analog of an extremely important class of eukaryotic reuptake proteins, solute carrier 6 (SLC6). Members of this class include the family of sodium-coupled transporters which hold a vital role in neurotransmission. These neurotransmitter sodium-coupled transporters use symport to couple sodium moving with its gradient with the reuptake of neurotransmitters from the synaptic cleft. This mechanism of reuptake occurs readily in serotonin and dopamine related synapses. Disorders of reuptake have long-reaching effects including complications of epilepsy, depression, and obsessive-compulsive disorder. Tricyclic antidepressants, and even illicit drugs such as cocaine can inhibit the proper functioning of these integral proteins (1). The difficulty in isolating and purifying these proteins from animal tissue lead to study of LeuT, instead, as a novel analog of this mundane class of animal symporters. Various tricyclic antidepressants were crystallized in complex with LeuT, including imipramine, clomipramine and desipramine (PDB = 2QB4) (6). The TCAs bound to an extracellular site 11 Angstroms above the substrate and outside of the extracellular gate. This binding stabilizes the occluded state of the protein, thus inhibiting the conformational rearrangement leading to the open-to-in state. A key difference between LeuT and the other SLC6 symporters such as the serotonin reuptake transporter is that the TCAs competitively inhibit protein activity. Necessarily, in these other symporters, the TCA binding site must overlap the binding site of the substrate molecules available for transport. For LeuT, the binding results in non-competitive inhibition. In the occluded state, there is competition between opening of the extracellular gate, or conformational opening of the intracellular gate involving rearrangement of the tertiary structure and breaking hydrogen bonds. It is postulated that TCAs help to latch off the extracellular gate through a salt bridge formed specifically with clomipramine, and other polar interactions with the other TCAs, thus slowing the rate of release of the extracellular gate, therefore slowing the unbinding of the substrate from its pocket. Another key difference between eukaryotic SLC6 members and LeuT is that SLC6 members are chloride dependent where LeuT operates independent of the chloride anion (4).
Bacterioferritin (PDB=2FKZ), isolated from Azotobacter vinelandii, is structurally similar to LeuT as designated by Dali Structural Alignment test. Dali utilizes the protein secondary structure in order to find proteins with similar protein structure and calculated a Z score from it. The higher the Z score, the more secondary structural similarities there are. Bacterioferritin is an iron transport protein which uses a heme protoporphyrin prosthetic group (8). Both bacterioferritin and LeuT contain a significant amount of alpha helices. These alpha helices mediate membrane transport due to their flexibility in protein structure and allowing easy conformational change of the protein structure. The Z score of structural similarity was 3.0. Looking at primary structure, the BLAST server analysis provides an analysis of the primary structure similarities between the target protein and a test protein, which is quantified in an E score. Minimization of the E score shows extreme structural similarities at the primary level. BLAST analysis of LeuT gave an interesting protein, Human Dihydropyrimidinease-2, which also is found in neurons. Human dihydroprimidinease-2 is responsible for the development and repair of axon termini and myelin sheath repair (7). The similarity between LeuT and Human dihydroprimidinease-2 can be related to their presence both in neurons and the neurological response. This suggests that there may be a physiological importance in specific sequences of proteins to characterization of their functions in neurons.