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

Renhong Yan,Jürg Gertsch,Yaning Li,Simon Singer,Karl-Heinz Altmann,Jennifer Müller,Xinyue Zhong,Qiang Zhou,Yuanyuan Zhang,Lu Xia

doi:10.1038/s41421-021-00247-4

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.

Highlights

L-type amino acid transporter 1 (LAT1, known as SLC7A5) mediates the pH-independent and sodiumindependent exchanging transport of the large neutral amino acids, thyroid hormones, and pharmaceutical drugs across the plasma membrane[1,2,3]
As a light chain protein, LAT1 is covalently linked with the heavy chain protein 4F2hc through a conserved disulfide bond, forming a heterodimeric LAT1-4F2hc complex that belongs to the heteromeric amino acid
JX-075, JX-078, and JX-119 did not induce measurable leucine efflux at a concentration of up to 100 μM, indicating that none of these compounds is a substrate for LAT1 (Supplementary Fig. S2)

Summary

Introduction

L-type amino acid transporter 1 (LAT1, known as SLC7A5) mediates the pH-independent and sodiumindependent exchanging transport of the large neutral amino acids, thyroid hormones, and pharmaceutical drugs across the plasma membrane[1,2,3]. The inward-open conformation cryo-EM structures of the human LAT1-4F2hc complex in its apo state or with the non-specific inhibitor 2-amino-2-norbornanecarboxylic acid (BCH) bound and some eukaryotic homologs of the HAT family were solved[12,13,14,15]. We solved the structure of the LAT1-4F2hc complex bound with Diiodo-Tyr. Structural comparison suggested that this structure might represent an intermediate conformation between the outward-facing occluded and outward-open states.

Results

Conclusion