Structural insights into thyroid hormone transport mechanisms of the L-type amino acid transporter 2

Abstract

Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3'-diiodothyronine (3,3'-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3'-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3'-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport.

Figure 1.. A, Crystal structure of the 12-TMH transporter AdiC, 3L1L (magenta), in the conformation of openward occluded state bound arginine (close up view, yellow). B, Homology model of the 12-TMH Lat2 light chain (green) and analogous binding site bound 3,3′-T₂ (orange). The amino acid moiety of arginine and accordingly of 3,3′-T₂ is bound to their recognition site, due to common fingerprint motifs (see Supplemental Figure 1), and is located between TMH1 and TMH6 via hydrogen bonds to the backbone.

Figure 2.. Mutation results of residues lining the potential TH-membrane-traversing channel in Lat2.

A, The Lat2 model indicates the putative locations of residues potentially involved in translocation of 3,3′-T₂ (orange) covering the potential channel in the center of Lat2. The color of the selected residues and mutations corresponds to the color of the uptake bar diagram. B, 3,3′-T₂ uptake (100nM) as percentage of wild type and normalized to the corresponding total expression of the different Lat2 variants that were coinjected with CD98 in X. laevis oocytes. Three different types of results are indicated; like wild type (gray), decreased (magenta) and increased 3,3′-T₂ uptake (cyan). Data are representative of at least 3 independent transport experiments. Significance level is indicated with *, P < .01; **, P < .001; ***, P < .0001.

Figure 3.. Lat2 homology model (green) bound 3,3′-T₂ (orange) within the traversing channel (gray).

A, Asparagines lining the TH-traversing channel, side chain shortening led to channel enlargement and increased the TH uptake. Potential gate function of Phe²⁴², here forming a lid covering 3,3′-T₂ in the bound openward-occluded state. B, Side chain enlargement of Tyr¹³⁰ to arginine obstructs the channel and results in a decrease in 3,3′-T₂ uptake. Phe²⁴² is interacts with the aromatic rings of 3,3′-T₂ via π-system stacking of the extracellular open state. Asymmetric excavations in the traversing channel allow bulky substituents either (C) at the tyrosine ring 3′,5′-T₂ or (D) at the phenyl ring for 3,5-T₂.

Figure 4.. Competitive inhibition of the transport by Lat2/CD98 using various substrates.

Amino acid-functional groups (A), proximate hydrophobic extension (B), and substituents in the 3 and 5 positions of the tyrosine ring (C) are studied. Transported substrates are highlighted in gray. Uptake of 3,3′-T₂ (100nM) in X. laevis oocytes coinjected with cRNA of CD98 and Lat2. Experiments have been performed in the absence (control) and presence of indicated substrates (A, 10μM; B and C, 1mM). Mean ± SEM of 10–15 oocytes per group of 2–3 independent experiments normalized to the uptake of 3,3′-T₂ (after subtraction of noninjected oocytes) are shown.

Figure 5.. Scheme of substrate translocation from the extracellular side routed through the center of Lat2.

Localizations are depicted of the recognition site (blue dashed) for carboxylic acid and amino group of the substrate (orange) and of a channel for substrate passage with asymmetric hollows. Arrangements of transport-sensitive residues are given and colored according to their mutational effects, eg, wild-type (gray), decreased (magenta), and increased 3,3′-T₂ uptake (cyan). F242 is suggested to be a switch site that potentially facilitates intracellular substrate release.

Figure 6.. Overlay of (A) BCH (yellow) with S12 (blue) and (B) 3,3′-T₂ (orange) with S7 (cyan) (see also Figure 4) docket into the binding pocket (gray surface) of Lat2, showing the allowed spatial dimensions of substrates to be transported.

Overlayed S12 (blue in A) is not transported, probably due to its large size (red arrow) that does not fit the binding pocket.

Figure 7.

Conformational flexibility search between the 2 aromatic rings of TH yielded the result that (A) 3,3′-T2 and (B) 3′,5′T₂ are flexible enough for uptake, whereas in C, T₄ steric hindrance of the bulky iodines restricts flexibility and prevents its uptake by Lat2.