Structural Studies of the Taurine Transporter: A Potential Biological Target from the GABA Transporter Subfamily in Cancer Therapy

Abstract

The taurine transporter (TauT, SLC6A6) is a member of the solute carrier 6 (SLC6) family, which plays multiple physiological roles. The SLC6 family is divided into four subfamilies: GABA (γ-aminobutyric acid), monoamine, glycine and neutral amino acid transporters. Proteins from the GABA group, including the taurine transporter, are primarily considered therapeutic targets for treating central nervous system disorders. However, recent studies have suggested that inhibitors of SLC6A6 could also serve as anticancer agents. Overexpression of TauT has been associated with the progression of colon and gastric cancer. The pool of known ligands of this transporter is limited and the exact spatial structure of taurine transporter remains unsolved. Understanding its structure could aid in the development of novel inhibitors. Therefore, we utilized homology modelling techniques to create models of TauT. Docking studies and molecular dynamics simulations were conducted to describe protein-ligand interactions. We compared the obtained information for TauT with literature data on other members of the GABA transporter group. Our in silico analysis allowed us to characterize the transporter structure and point out amino acids crucial for ligand binding: Glu406, Gly62 and Tyr138. The significance of selected residues was confirmed through structural studies of mutants. These results will aid in the development of novel taurine transporter inhibitors, which can be explored as anticancer agents.

Keywords: SLC6A6; cancer; docking; homology modelling; molecular dynamics; structure; taurine transporter.

Figure 1

Overexpression of GABA transporters is associated with several types of cancer.

Figure 2

Topology diagram of the transporter from SLC6 family—spatial structure of SLC6A1 (GAT1) transporter with selected residues (left panel). Residues color: blue—S2 site, yellow—extracellular gate, green—S1 site, and pink—intracellular gate. Schematic representation mechanism of action of the transporter (right panel). Blue sphere—substrate; yellow sphere—ions.

Figure 3

Spatial structure of SLC6A6 transporter. Yellow cloud marks the ligand binding site.

Figure 4

The overall structure of the taurine transporter in an inward-occluded state (left panel). Superposition of SLC6A6 model and GAT1 structure (PDB code: 7Y7W). View on the Ser464 in the TM10 domain from SLC6A6 (orange stick and cartoon), TM10 from GAT1 (green cartoon) (right panel).

Figure 5

Schematic representation of taurine transporter conformational changes during transport: outward-open conformation (left panel), occluded states (middle panel), inward-open conformation (right panel). Occluded states represent average structure for outward-open and inward-open conformation, due to small-distance changes in domains and residues between each other. Domains shown as cartoons, colored as follows: TM1—dark blue, TM2—marine, TM3—light blue, TM6—green, TM8—yellow, and TM10—orange. Yellow sticks—residues from the extracellular gate, pink sticks—residues from the intracellular gate, and purple—taurine. For clarity, TM11, TM12, ions and ligands are not shown.

Figure 6

Electrostatic potential maps for the S1 binding site of taurine (left panel) and creatine (right panel) transporters. Red color—accumulation of negative electrostatic potential; blue color—accumulation of positive electrostatic potential.

Figure 7

Electrostatic potential maps for the S1 binding site of taurine transporter mutants: Gly58Glu (left, upper panel), Leu306Gln (right, upper panel), Glu406Gly (left, down panel), and Glu406Cys (right, down panel). Red color—accumulation of negative electrostatic potential; blue color—accumulation of positive electrostatic potential.

Figure 8

Structures of taurine transporter substrates [37].

Figure 9

The overall structure of SLC6A6 with substrate—upper panel. Different colors of taurine represent its localization in: outward-open (blue), outward-occluded (green), inward-occluded (yellow), and inward-open (orange) states. Taurine binding mode within TauT: Outward-open state—middle, left panel (template: 4MMB, tool: SwissMODEL), outward-occluded state—middle, right panel (template: 2Q6H, tool: SwissMODEL), inward-occluded state—down, left panel (template: 7Y7W, tool: Modeller), and inward-open state—down, right panel (template: 7Y7Z, tool: Modeller). Residues color: yellow—extracellular gate, green—S1 site, blue sphere—sodium ion, and taurine—purple.

Figure 10

Binding mode of further substrates in an inward-occluded state of the taurine transporter (template: 7Y7W, tool: Modeller): hypotaurine (left, upper panel), β-alanine (right, upper panel), and GABA (left, down panel). For comparison, position of GABA in human GAT1 is shown (PDB code: 7Y7W; right, down panel). Residues color: yellow—extracellular gate, green—S1 site, blue sphere—sodium ion, red sphere—water molecules, hypotaurine—brown, β-alanine—marine, and GABA—slate.

Figure 11

Structures of selected taurine transporter inhibitors. Their biological activity was determined by [³H]-taurine uptake using HEK293 cells producing TauT-GFP (green fluorescent protein) and expressed as IC₅₀ values [37].

Figure 12

Binding mode of selected inhibitors in an inward-occluded state (template: 7Y7W, tool: Modeller) of the taurine transporter—P4S (left panel) and I4AA (right panel). Residues color: yellow—extracellular gate, green—S1 site, blue sphere—sodium ion, P4S—blue, and I4AA—green.

Figure 13

Distance changes between inhibitors and crucial amino acids: P4S (left, upper panel), I4AA (right, upper panel). Interactions color: orange; Glu406-COOH—ligand, yellow; Gly62-NH—ligand, green; Tyr138-OH—ligand. RMSD changes for protein and inhibitors: P4S (left, down panel) and I4AA (right, down panel) during molecular dynamics.

Figure 14

Binding mode of taurine within SLC6A6 mutants. Models were built based on the 4MMB template with SwissMODEL. Glu406Gly mutant (left panel); Glu406Cys (right panel). Residues color: yellow—extracellular gate, green—S1 site, blue sphere—sodium ion, and taurine—purple.

Figure 15

Binding mode of taurine within SLC6A6 mutants. Models were built based on the 4MMB template with SwissMODEL. Gly57Glu (left panel); Leu306Gln (right panel). Residues color: yellow—extracellular gate, green—S1 site, blue sphere—sodium ion, and taurine—purple.