Substrate and drug binding sites in LeuT

Abstract

Summary of recent advances LeuT is a member of the neurotransmitter/sodium symporter family, which includes the neuronal transporters for serotonin, norepinephrine and dopamine. The original crystal structure of LeuT shows a primary leucine binding site at the center of the protein. LeuT is inhibited by different classes of antidepressants that act as potent inhibitors of the serotonin transporter. The newly determined crystal structures of LeuT-antidepressant complexes provide opportunities to probe drug binding in the serotonin transporter, of which the exact position remains controversial. Structure of a LeuT-tryptophan complex shows an overlapping binding site with the primary substrate site. A secondary substrate binding site was recently identified, where the binding of a leucine triggers the cytoplasmic release of the primary substrate. This two binding site model presents opportunities for a better understanding of drug binding and the mechanism of inhibition for mammalian transporters.

Structure-based discovery of prescription drugs that interact with the norepinephrine transporter, NET

“…No structures of human SLC6 members have been determined at atomic resolution; however, the leucine transporter LeuT from the bacterium Aquifex aeolicus has been determined by X-ray crystallography in different conformations, with various ligands, including substrates and inhibitors (6). Additional crystallographic and model structures of related transporters (7)(8)(9)(10)(11) revealed that the SLC6 family members transport ligands across the cell membrane via the "alternating access" transport mechanism (12,13).…”

“…In particular, for a substrate to be transported efficiently, it needs to bind to the binding site on the transporter surface as well as fit within the binding cavity of the "occluded" transporter state (14); in contrast, a competitive inhibitor binds to the binding site, but is too large to fit into the binding cavity of the occluded state and thus is not transported through the transporter. In addition to the primary binding site (S1 site), the family members may have additional binding sites such as the clomipramine-binding site on LeuT (6,15).…”

High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

“…For example, the heme precursor 5-ALA is involved in the development of the neurological symptoms of porphyria (70,71). Medical uses of 5-ALA include the photodynamic detection of various tumors (especially in the CNS) and its use as a photosensitizer for photodynamic therapy of many diseases (11,72,73). Nevertheless, to date it is not fully understood how 5-ALA crosses the BBB.…”

“…No structures of human SLC6 members, including GAT-2, have been determined at atomic resolution; however, x-ray structures of a bacterial homolog, the leucine transporter LeuT, have been determined in four different conformations that were proposed to represent different snapshots of the transport cycle (11)(12)(13). Additionally, LeuT complex structures with various substrates and inhibitors suggested a competitive inhibition mechanism in which larger ligands (e.g., tryptophan) stabilize an inhibited outward facing conformation (12).…”

“…Additionally, LeuT complex structures with various substrates and inhibitors suggested a competitive inhibition mechanism in which larger ligands (e.g., tryptophan) stabilize an inhibited outward facing conformation (12). Interestingly, it was also shown that an additional substrate-binding site (S2) is located on the surface of LeuT (14 -16) and that various inhibitors stabilize different conformations in LeuT via sites overlapping with the S2 site (11,17). These observations are in agreement with the notion that LeuT and its human homologs transport ligands across the cell membrane via the "alternating access" transport mechanism (13, 19 -25).…”