Molecular dynamics insights into structural and hydration dynamics of LAT1 bound to ligands and JPH203-induced inward-open arrest

L-type amino acid transporter 1 (LAT1) delivers amino acids and amino acid-mimicking drugs across blood–brain barrier and is a key underexplored target against cancer. We investigated molecular triggers of LAT1 conformational changes upon ligand binding performing molecular dynamics simulations on LAT1-substrates and one inhibitor. We realized LAT1 conformational change occurs via a two-step expansion-contraction of the mid-section and water flow propels substrate translocation. Expansion allows water flow from the extracellular H6/H10 sub-pocket, facilitating ligand reorientation, while contraction promotes ligand movement toward H8 and water flow toward the intracellular region. For large substrates (cpd1), leaving the H6/H10 sub-pocket toward H10–H3 is coupled to mid-section expansion of the transporter, promoting water flow and ligand reorientation that drive H10 rotation and intracellular passage. Furthermore, benzoxazole tail of JPH203, a clinically investigated LAT1 inhibitor, can rotate downward during expansion phase, arresting LAT1 in the inward-open conformation by displacing unfavorable water molecules. This finding extends JPH203’s original mechanism, showing that it can block LAT1 not only in outward-facing conformation but by stabilizing the inward-open state. L-type amino acid transporter 1 (LAT1) is a promising yet underexplored target against cancer, with current understanding of LAT1 inhibition being largely based on static structures of LAT1–inhibitor complexes. Here, the authors study molecular triggers of LAT1 conformational changes upon ligand binding through molecular dynamics simulations on LAT1-substrates and one inhibitor, JPH203, revealing alterations in hydration dynamics during transport and showing that JPH203 can block LAT1 not only in an outward-facing conformation but by stabilizing the inward-open state.