ECD of PepT1 interacts with TM1 to facilitate substrate transport

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of a wide variety of di- and tri-peptides and are essential for the absorption of dietary peptides in the digestive tract and the recovery of peptides in renal filtrate. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has 12 transmembrane (TM) helices that fold into two domains, the N-terminal domain (NTD, TM1-6) and C-terminal domain (CTD, TM7-12), and a large extracellular domain (ECD) of ∼200 amino acids between TM9 and TM10. It is known that peptide transport involves large motions of the N- and C-domains, but the role of ECD remains unclear. Here we report the structure of PepT1 from Equus caballus (horse) determined by cryo-electron microscopy. The structure shows that ECD interacts with TM1 and bridges the N- and C-domains. Deletion of the ECD or mutations to the TM1-ECD interface both impair the transport activity. These results demonstrate a role of ECD in structure and function of PepT1 and enhance our understanding of the mechanism of transport in PepT1.