User:Neha Bang/Sandbox 1
From Proteopedia
Contents |
Leucine Transporter
Introduction
LeuT is a membrane transporter naturally found in certain prokaryotes and a member belonging to the SLC6 family of transporters (1). These proteins couple the transport of solutes against their gradients to the co-transport of sodium and chloride with their gradients (2). Many of these transporters participate in the reuptake of neurotransmitters such as dopamine, serotonin, and norepinephrine. For example, the transporter DAT couples the transport of dopamine to the co-transport of Na+ ions. Chloride ions may be used to increase the binding affinity of Na+ ions to the transporter (2, 10, 11, 13). The dysfunction of this protein can contribute to disorders such as Parkinsonism, Tourette syndrome, substance abuse, and ADHD (2). SERT, another member of the SLC6 family, requires the co-transport of Na+ and Cl- ions, as well as the counter transport of K+ ions to transport serotonin into tissues of the central and peripheral nervous system, as well as epithelial cells and platelets (2, 12).
Mechanism
Two Na+ ions bind to LeuT to facilitate substrate binding. The first Na+ ion binds to the Na2 site when the protein is facing the inside of the cell. This causes LeuT to convert to the open-to-out state. Glu-290, a residue near this site, gets deprotonated, widening the extracellular vestibule (4, 5). This gate, comprised of residues R30 and D404, controls the entry of small organic substrates to LeuT. The presence of a wider gate makes LeuT more accessible to the other Na+ ion. The second Na+ ion binds to the Na1 site near the binding pocket. The binding of both Na+ ions causes transmembrane domains 1 and 6 (TM1 and TM6) to rearrange, thereby widening the extracellular gate. This makes it easier for the substrate to access LeuT from the outside of the cell (3, 4, 8, 9). Both ions remain when the substrate arrives at the gate.
The substrate then binds to the extracellular vestibule (1). Residues R30 and D404 bind to each other via electrostatic interactions, thereby closing the gate (1). Residues F253 and Y108 interact with each other and align above the substrate. This blocks the entry of other substrates to LeuT (1). Then the substrate travels to and binds to the primary binding site (1). The binding of sodium at the Na1 site contributes to a favorable binding free energy between the substrate and LeuT (7). This makes it more likely for the substrate to bind tightly enough to the protein to get transported. This ion participates in an ionic interaction with the carboxylate group of the substrate, thereby stabilizing the substrate (4, 5, 6).
The interactions of Na+ and the residues R30, D404, F253, and Y108 promote the formation of the occluded state, in which the protein closes around the substrate, trapping it inside its active site. The binding pocket is closed to both sides of the membrane (3). Once the pocket has closed, multiple interactions secure the substrate in the binding pocket, including a hydrogen bond formed between the hydroxyl of Y108 and the substrate. These interactions seal the binding pocket, closing it to either side of the membrane (3, 5).
LeuT must go through multiple conformational stages in order to switch from the occluded to the open-to-in conformation (3). TM 1b rotates outwards (3). A glycine-rich loop adds flexibility to allow LeuT to change to different intermediate conformations (1, 3). These interactions allow the binding site to become compressed and, therefore, to change the conformation of the protein to the open-to-in state (1). This allows LeuT to release the substrate into the cell.
References
1. Gouaux, Eric, Piscitelli, L. Chayne, Singh, K. Satinder, Yamashita, Atsuko. A Competitive Inhibitor Traps LeuT in an Open-to-Out Conformation. Science. 2008; 322: 1655-1660. Doi: 10.1126/science. 1166777.
2. N. H. Chen, M. E. Reith, M. W. Quick, Pflugers Arch. 447, 519 (2004).
3. B. I. Kanner, J. Membr. Biol. 213, 89 (2006).
4. Gracia, L., Noskov, S., Shi, L., Stolzenberg, S., Weinstein, H., Zhao, C. Ion-Controlled Conformational Dynamics in the Outward-Open Transition from an Occluded State of LeuT. Biophy Journal. 2012; 103(5): 878-888. Doi: 10.1016/j.bpg.2012.07.044.
5. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., Gouaux, E. 2005. Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437: 215-223.
6. Kanner, B. I. 2005. Molecular physiology: intimate contact enables transport. Nature 437: 203-205.
7. Noskov S.Y., Roux B. Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms. J. Mol. Biol. 2008; 377:804–818.
8. Zhao Y., Terry D., Javitch J.A. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature. 2010;465:188–193.
9. Krishnamurthy H., Gouaux E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature. 2012; 481: 469–474.
10. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., Gouaux, E. 2005. Crystal Structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437: 215-223.
11. Bossi, E., Giovannardi, S., Binda, F., Forlani, G., Peres, A. 2002. Role of Anion-Cation Interactions on the Pre-Steady-State Currents of the Rat Na+/Cl--Dependent GABA Cotransporter rGAT1. J. Physiol (Lond) 541: 343-350.
12. Loo, DD., Eskandari, S., Boorer, K.J., Sarkar, H. K., Wright, E.M. 2000. Role of Cl- in Electrogenic Na+-Coupled Cotransporters GAT1 and SGLT1. J. Biol. Chem. 275: 37414-37422.
13. Rudnick, G., Clark, J. 1993. From Synapse to Vesicle: the Reuptake and Storage of Biogenic Amine Neurotransmitters. Biochim Biophy. Acta. 1144: 249-263.
