Structural mechanisms of B0AT1 substrate recognition and complex assembly

Amino acid transporters are essential for maintaining cellular amino acid homeostasis. The broad-specificity neutral amino acid transporter B 0 AT1 is predominantly expressed in the intestine and kidney, where it mediates the uptake and reuptake of neutral amino acids with varying affinities. Proper plasma-membrane localization of B 0 AT1 requires dimerization with angiotensin-converting enzyme 2 (ACE2) in the intestine or collectrin in the kidney. Structural information is available for apo and substrate-bound B 0 AT1-ACE2 complexes, but how B 0 AT1 interacts with different accessory proteins and recognizes smaller substrates remains enigmatic. Here, we compare cryo-EM structures of B 0 AT1 in complex with ACE2 or collectrin in substrate-free states, and bound to alanine, leucine or tryptophan. The complexes display only minor differences in accessory-protein binding through a conserved interaction surface. Substrate recognition follows a canonical architecture observed for other SLC6 family transporters, with subtle adaptations that potentially account for the different affinities of the three amino acids. Notably, we resolve several cholesterol molecules surrounding the transporter, highlighting a potential regulatory role of the membrane environment that we address by molecular dynamics simulations.