Structural basis of substrate recognition and allosteric inhibition in human B ⁰ AT2

The SLC6 family is a major target for neuropsychiatric therapeutics. Human B ⁰ AT2 (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 B ⁰ AT2 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 B ⁰ AT2 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.