Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter

Abstract

LAT1 (SLC7A5) is one of the representative light chain proteins of heteromeric amino acid transporters, forming a heterodimer with its heavy chain partner 4F2hc (SLC3A2). LAT1 is overexpressed in many types of tumors and mediates the transfer of drugs and hormones across the blood-brain barrier. Thus, LAT1 is considered as a drug target for cancer treatment and may be exploited for drug delivery into the brain. Here, we synthesized three potent inhibitors of human LAT1, which inhibit transport of leucine with IC₅₀ values between 100 and 250 nM, and solved the cryo-EM structures of the corresponding LAT1-4F2hc complexes with these inhibitors bound at resolution of up to 2.7 or 2.8 Å. The protein assumes an outward-facing occluded conformation, with the inhibitors bound in the classical substrate binding pocket, but with their tails wedged between the substrate binding site and TM10 of LAT1. We also solved the complex structure of LAT1-4F2hc with 3,5-diiodo-L-tyrosine (Diiodo-Tyr) at 3.4 Å overall resolution, which revealed a different inhibition mechanism and might represent an intermediate conformation between the outward-facing occluded state mentioned above and the outward-open state. To our knowledge, this is the first time that the outward-facing conformation is revealed for the HAT family. Our results unveil more important insights into the working mechanisms of HATs and provide a structural basis for future drug design.

Fig. 1. Overall structure of the LAT1-4F2hc bound with JX-078.

a Chemical formulas of the designed LAT1 inhibitors. b The overall cryo-EM map of the LAT1-4F2hc complex bound with JX-078. c The overall structure of the LAT1-4F2hc complex bound with JX-078. The glycosylation moieties are shown as sticks. H, helix. 4F2hc and LAT1 are colored orange and deep blue, respectively.

Fig. 2. Inhibitor binding analysis.

a JX-078 binding mode in LAT1. b The hydrophobic interactions around the tail of JX-078. c LAT1 has a broad extracellular vestibule above JX-078. d Comparison of the binding mode of the three inhibitors. JX-075, JX-078, and JX-119 are colored green, yellow, and red, respectively.

Fig. 3. Overall structure of the LAT1-4F2hc bound with Diiodo-Tyr.

a The overall structure of the LAT1-4F2hc bound with Diiodo-Tyr. The structure on the left is the EM map of the complex. The structure on the right is the overall structure of the complex. The glycosylation moieties are shown as sticks. H, helix. 4F2hc and LAT1 are colored orange and blue, respectively. b Diiodo-Tyr binding mode in LAT1. c LAT1 has a broad extracellular vestibule above Diiodo-Tyr.

Fig. 4. Conformational change between inward-open and outward-occluded state.

a Comparison of the outward-occluded structure of LAT1 and the inward-facing structure of LAT1 (PDB ID: 6IRT). The core domain of the outward-occluded LAT1 rotates for ~27° compared with that of inward-open LAT1. The interaction mode at the extracellular interface of the LAT1-4F2hc complex is different between the inward-facing and the outward-occluded conformations. The red box corresponds to b. b The details of the extracellular interface and the polar interactions are shown as red dashed lines for the outward-occluded conformation and gray dashed lines for the inward-facing conformation. The new residues in the outward-occluded conformation are labeled in red. c The hydrogen bond network at the intracellular side of the outward-facing conformation. d The hydrophobic interactions stabilize the outward-facing conformation with the hydrophobic residues shown as spheres.

Fig. 5. The asymmetrical movement of TM1 and TM6.

a Comparison between the outward-occluded structure of LAT1 bound with JX-078 and that bound with Diiodo-Tyr, with the side view, the bottom view, and the top view shown in left, middle, and right panel, respectively. b Structural comparison among the inward-facing structure of LAT1 bound with BCH and the outward-occluded structure of LAT1 bound with JX-078 or Diiodo-Tyr (left panel). The movement of TM1 and TM6 is shown in the middle and right panel, respectively. BCH, JX-078, and Diiodo-Tyr are colored pink, yellow, and lemon green, respectively.

Fig. 6. Putative working model for the LAT1-4F2hc complex.

The model shows a transport mechanism of LAT1. LAT1 loads substrates in cytoplasm and then TM1a and TM6b rotate to the hash domain to close the inward gate. During the transition from the inward-facing conformation to the outward-facing conformation, TM1a and TM6b continue to rotate and TM1b and TM6a start to rotate away from the hash domain. Then TM1b and TM6a undergo further rotation and push the gating residue Phe252 away from the occluded configuration, triggering substrate release. For simplicity, TM2 and TM7 of the core domain and TM8 of the hash domain are not shown here. ECD, extracellular domain. H, helix. TM, transmembrane helix.