Towards a Structural Understanding of Alternating Access within the Proton Dependent Oligopeptide Transporter (POT) Family

Abstract

Recently we determined the first crystal structure of a member of the Proton dependent Oligopeptide Transporter (POT) family, PepTSo, a prokaryotic homologue of the mammalian PepT1 and PepT2 proteins. PepT1 and PepT2 are responsible for the uptake and re-absorption of dietary peptides and have a direct effect on the oral bioavailability of important drug families. POT family transporters belong to the Major Facilitator Superfamily (MFS) of secondary active transport proteins and contain between 12 and 14 transmembrane helices. The structure of PepTSo revealed a ligand bound occluded state that was noticeably asymmetric between the two canonical N- and C-terminal six helical bundles present in MFS members. To gain further insight into the transport mechanism of the POT family we have determined the structure of a second member to 3.3 Angstrom resolution. Compared to PepTSo, this second structure reveals an essentially symmetric inward open conformation. Here we present the analysis of this structure and show that using previously identified inverted topology repeats present within the MFS fold we can create a model for the currently missing outward open conformation. We further provide experimental evidence for the outward open model using double electron electron resonance (DEER) measurements. These two crystal structures, combined with the repeat swapped model, essentially describe the basic set of conformations required for the alternating access model of transport within this pharmaceutically important family of MFS transporters.