Abstract

The proton-coupled amino-acid transporter SLC36A1 (hPAT1) is an atypical H⁺-driven carrier and mediates the intestinal absorption of a wide array of zwitterionic amino-acid analogs, including many compounds with central nervous-system (CNS) activity, as well as the activation of the mTORC1 pathway and the export of amino acids from lysosomes, thereby maintaining cellular amino-acid homeostasis. Here, we present the cryo-EM structures of a member of the SLC36 family, hPAT1, in its apo state and in complex with three chemically distinct substrates, including the α-amino acid D-serine, the β-amino acid nipecotic acid, and the heterocyclic drug D-cycloserine, at resolutions of 3.4–3.5 Å. Despite their chemical diversity, all ligands adopt a spatially convergent binding mode, elucidating the structural basis for PAT1’s broad substrate promiscuity. In addition, we identify E270 as a potential proton-binding site. Together, these findings provide structural insights into the molecular mechanism of proton-coupled amino acid transport. Notably, the cryo-EM structure of PAT1 bound to D-cycloserine illustrates a viable oral CNS drug delivery strategy: exploiting polar scaffolds to achieve transporter-mediated intestinal absorption and blood–brain barrier penetration without relying on high lipophilicity.