Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

Amino acids play a central role in cellular metabolism. Dysregulation of both intra- and extracellular amino acid concentrations is associated with pathological conditions (1). Amino acid transfer across the plasma membrane is mediated by specific transporters that bind and transport these molecules from the extracellular medium into the cell or vice versa.Heteromeric amino acid transporters (HATs) are a family of amino acid transporters comprised by a heavy subunit and a light subunit, linked by a conserved disulfide bridge (2). Heavy subunits (SLC3 family) are ancillary proteins required for trafficking the holotransporter to the plasma membrane (2), whereas the light subunits (LATs; SLC7 family) transport amino acids and confer substrate specificity to the heterodimer (2). HATs are amino acid exchangers that harmonize amino acid concentrations at each side of the plasma membrane and as such they play a critical role in amino acid homeostasis (1, 3).The physiological relevance of HATs is highlighted by their role in cancer and several inherited diseases (4–8). HAT neutral amino acid transporters in particular are gaining momentum as several mutations linked to human diseases have recently been identified, and new physiological roles for this group of transporters have been uncovered using knockout mouse models (8–13). Several loss-of-function mutations in human LAT2/CD98hc (SLC7A8/SLC3A2) are associated with age-related hearing loss (ARHL) (9) and cataracts (10). Also, some coding variants are linked to an increased risk of autism spectrum disorder (14). In addition, hLAT2/CD98hc overexpression in pancreatic cancer cells sustains glutamine-dependent mTOR activation to promote glycolysis and chemoresistance (15). This observation thus points to hLAT2/CD98hc as a potential pharmacological target in this particular type of cancer. On the other hand, LAT1/CD98hc (SLC7A5/SLC3A2), which is also linked to cancer (4, 7), participates in brain development and autism spectrum disorder (12). Finally, Asc1/CD98hc (SLC7A10/SLC3A2) is considered a target to regulate glutamatergic neurotransmission in some cognitive disorders, such as schizophrenia (16, 17), and a relevant player in adipocyte lipid storage, obesity, and insulin resistance (18).Several atomic structures of HATs (19–24) and LATs (25) have recently been described, thus paving the way for the dissection of the molecular transport mechanisms. The substrate-binding site of LATs determined in complex with a substrate or competitive inhibitors shows a conserved design consisting of two unwound segments of transmembrane (TM) 1 and TM6, which contain residues that recognize the α-amino and carboxyl groups of the substrate (21–25). Each member of the HAT family displays a preference for transporting a certain set of substrates (2). LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc transport neutral amino acids but of different sizes. LAT1 is specialized in large neutral amino acids but it is inefficient for L-glutamine, and it does not transport small amino acids. LAT2 transports both large and small neutral amino acids, and it is highly efficient for L-glutamine. Finally, Asc1 mediates the preferential uptake of small neutral amino acids, including D-isomers, particularly D-serine (26–28).Despite recent advances in resolving the structure of several HATs, the molecular mechanisms explaining why each member of the family shows exquisite preference for certain substrates but not others are mostly unknown. Here, we addressed the structural bases of substrate specificity in the HAT family. To this end, we used cryo–electron microscopy (cryo-EM) to determine the structure of human LAT2/CD98hc in inward-facing open and apo conformation. We used this structure to study substrate-binding determinants by combining Protein Energy Landscape Exploration (PELE) and molecular dynamics (MD), together with mutational and functional studies. We reveal that a few residues present in the substrate-binding pocket and nearby regions determine substrate preference, and we demonstrate how the substrate preference of several HATs can be interconverted. In addition, a region located at a certain distance of the substrate cavity but whose structure critically influences the conformation of the substrate-binding site also regulates substrate preference. This region accumulates mutations associated with ARHL and cataracts that alter hLAT2 substrate specificity.Our work uncovers key structural determinants that govern, by different mechanisms, the differences in substrate specificity found within HAT members of neutral amino acid transporters. It also provides the structural bases for mutations in LAT2/CD98hc associated with deafness and cataracts.