In human and other mammalian cells, transport of L-lactate across plasma membranes is mainly catalyzed by monocarboxylate transporters (MCTs) of the SLC16 solute carrier family. MCTs play an important role in cancer metabolism and are promising targets for tumor treatment. Here, we report the crystal structures of an SLC16 family homologue with two different bound ligands at 2.54 and 2.69 Å resolution. The structures show the transporter in the pharmacologically relevant outward-open conformation. Structural information together with a detailed structure-based analysis of the transport function provide important insights into the molecular working mechanisms of ligand binding and L-lactate transport.

Significance: Here we report the structural bases of the interaction between the catalytic light subunit and the heavy subunit of the heteromeric amino acid transporters. Our data show that the large ectodomain of the human heavy subunit 4F2hc plays a key role in the interaction and stability of the light subunit L-type amino acid transporter 2. This finding paves the way for structural and functional studies to elucidate the role of the heavy subunit in the regulation of these transporters; such studies will be highly relevant in human pathology.

Significance: Disease-causing bacteria are able to survive the strongly acidic environment of the stomach by activating extreme acid-resistance responses. One of these responses in gut bacteria consists of converting l -arginine into agmatine, which results in removal of one proton from the cytoplasm. In Escherichia coli , the transport protein AdiC assures the efflux of agmatine in exchange with l -arginine. We have solved the structures of wild-type AdiC in the presence and absence of the substrate agmatine at high resolution, allowing for the identification of crucial water molecules and of their functional roles in the substrate-binding pocket. Furthermore, structure-based site-directed mutagenesis combined with a radioligand binding assay improved our understanding of substrate binding and specificity of the l -arginine/agmatine antiporter AdiC.

Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1’s substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.