Structural basis of substrate recognition and allosteric inhibition in human B0AT2

The SLC6 family is a major target for neuropsychiatric therapeutics. Human B0AT2 (SLC6A15) regulates cerebral amino acid homeostasis and glutamatergic transmission and has been linked to major depressive disorder, yet its transport and inhibition mechanisms remain unclear. Here we report cryo-EM structures of human B0AT2 in the apo state and in complex with three substrates (proline, leucine, and methionine) and two inhibitors (loratadine and tiagabine), capturing outward-open, early substrate-bound intermediate, outward-occluded, and inward-open conformations along the transport cycle. These structures reveal a local conformational tuning at the canonical substrate-binding pocket (S1), in which rearrangement of Phe308 remodels the pocket geometry to tune substrate accommodation and selectivity. Loratadine stabilizes an outward-occluded state via allosteric inhibition at the extracellular S2 pocket, whereas tiagabine stabilizes the inward-open state through cooperative multi-site inhibition involving the S1 site and two previously unrecognized intracellular cavities (S3 and S4). Together with functional assays and mutagenesis, these data define the molecular basis of B0AT2 substrate selectivity and state-dependent inhibition. Notably, the two newly identified intracellular cavities are broadly conserved within the SLC6 family, reflecting a common intracellular vestibular architecture and enabling the rational design of conformation-selective modulators for neuropsychiatric disorders.