LAT1, a pivotal actor on cancer stage: when function meets structure

LAT1 (SLC7A5) is a Na+ and pH-independent amino acid antiporter that regulates distribution of His and some large neutral amino acids, involved in amino acid metabolism, across cell membranes. LAT1 belongs to a special group of Heterodimeric Amino-acid Transporters (HATs) structurally coupled to the glycoprotein CD98 (SLC3A2). LAT1 is ubiquitous and it is also over-expressed in many human cancers that are characterized by increased demand of amino acids and it was recently acknowledged as a novel target for cancer therapy. The knowledge on molecular mechanism of LAT1 transport is still scarce, but some elegant studies on other related transporters report some transport mechanism connected both with 12 α-helices transmembrane symmetry and their structural rearrangement mediated by electrostatic interactions with some pivotal gating residues. For these reasons, the homology structural model of hLAT1 has been proposed using the bacterial AdiC transporter, which really showcases its gating symmetry. In the present work, LAT1 homology models in different conformational states were constructed via homology modelling procedures and tested through molecular dynamic simulation to predict amino acid residues critical for substrate recognition and translocation. His and the other amino-acids were docked in the LAT1 binding site to study both gating regions and the exposed residues involved in neutral amino-acid transport. Interestingly, some of these residues correspond to those previously hypothesized by literature and a novel one was identified. Finally, to validate the in silico structural evidence, the identified residues were tested in vitro through transport assay on different LAT1 mutants. For the first time, we demonstrated the crucial role in neutral amino acids transport of these residues by combined approaches of bioinformatics, site-directed mutagenesis, chemical modification, and transport assay in proteoliposomes. This novel information unravels dark sides of human LAT1 structure/function relationships and it may have also important outcomes in cancer novel inhibitors design of LAT1-mediated transport.