Recent advances in understanding proton coupled peptide transport via the POT family

Abstract

The POT family of membrane transporters use the inwardly directed proton electrochemical gradient to drive the uptake of essential nutrients into the cell. Originally discovered in bacteria, members of the family have been found in all kingdoms of life except the archaea. A remarkable feature of the family is their diverse substrate promiscuity. Whereas in mammals and bacteria they are predominantly di- and tri-peptide transporters, in plants the family has diverged to recognize nitrate, plant defence compounds and hormones. This promiscuity has led to the development of peptide-based pro-drugs that use PepT1 and PepT2, the mammalian homologues, to improve oral drug delivery. Recent crystal structures from bacterial and plant members of the family have revealed conserved features of the ligand-binding site and provided insights into post-translational regulation. Here I review the current understanding of transport, ligand promiscuity and regulation within the POT family.

Figure 1. Structure of POT family transporters and transport mechanism.

A. Crystal structure of PepT_So (PDB:2XUT) showing the core 12 TM domains arranged equally into an N- and C-terminal bundle and the additional helical hairpin, HA & HB, inserted between TM6 and TM7. B. Current model of proton coupled transport by the POT family. Transport is predominantly controlled through the opening and closing of two gates that sit on either side of a central binding site. The gates are made helices 1,2,4,5 from the N-terminal bundle and 7,8,10,11 from the C-terminal bundle. In many POT family members salt bridge interactions are seen coordinating the open/closed state of the gates. Proton binding/release from conserved proton binding sites act to drive the transport cycle in the forward direction, thereby concentrating peptides inside the cell.

Figure 2. Specificity pockets coordinate peptide binding.

A. Crystal structure of PepT_St shown in the plane of the membrane with the di-peptide Ala-Phe (PDB: 4D2C) (yellow) and tri-peptide Ala-Ala-Ala (PDB: 4D2D) (orange) shown as sticks. B. Close up view of the specificity pockets formed around the Ala-Phe peptide. C. Schematic detailing the interactions made to the Ala-Phe peptide shown in B. D. Equivalent view of B in PepT_So2 (PDB: 4TPJ), showing the specificity pockets identified in this POT family transporter. The Ala-Phe peptide from PepT_St is shown overlaid in grey.

Figure 3. Peptide can adopt different conformations in the binding site.

A. Crystal structure of PepTSt bound to tri-peptide Ala-Ala-Ala (orange). The specificity pockets are made predominantly from TM1 and TM7 and differ slightly between the di-peptide AlaPhe (yellow). B. Overlay of the peptides bound to PepTSt and PepTSo2. For clarity only the AAA peptide is shown for PepTSo2. Arrows indicate similar directions for the side chains. C. Steady state uptake of di- and tri-alanine by PepTSt under the same proton electrochemical gradient. The increased uptake of di-ala confirming the dual stoichiometry transport mechanism in this POT transporter.

Figure 4. Eukaryotic POT family transporters.

A. Crystal structure of NRT1.1 bound to nitrate (PDB: 5A2O & 4OH3). The binding pocket for nitrate is shown coloured according to the electrostatic properties of the side chains. The principle nitrate binding residue, H356, is shown. The intracellular gate helices, TM 4 & 5 are coloured cyan, with Thr101 shown as sphered in magenta. B. Close up view of the position of Thr101 in relation to the intracellular gate. C. Crystal structure of the extracellular domain of rat PepT2 (PDB:5A9H), showing overall dimensions. D. Hybrid model of human PepT1 showing the location of the extracellular and trans membrane domains. A model of trypsin has been docked in the putative interaction site.