Proton movement and coupling in the POT family of peptide transporters

Abstract

POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.

Keywords: biophysics; major facilitator superfamily; membrane transport; peptide transport; proton movement.

Fig. 1.

Residues important for proton coupling in PepT_So. (A) Proton-driven and counterflow data for conserved residues in PepT_So. (B) Structure of PepT_So in an inward-open conformation (PDB ID code 4UVM) indicating the location of residues involved in proton coupling and their interacting partners and highlighting the extracellular cavity leading to His61. (C) Rate of proton driven transport of both WT and the His61Asp variant at different pH values.

Fig. 2.

PepT_So transports both di- and trialanine using the same number of protons. Steady-state accumulation of di- and trialanine, driven using a fixed ΔμH⁺, in PepT_St (A) and PepT_So (B, solid lines). Dashed lines indicate the steady-state accumulation of di-and trialanine driven using ΔpH only. Schematics show the experimental setup; the gray triangle indicates a ΔpH, alkaline inside produced from an acetate diffusion gradient and − indicates a negative inside membrane potential produced through a potassium gradient.

Fig. 3.

Conservation of the TM2 histidine in mammalian and mammalian-like POT family transporters. (A) The extracellular cavity from PepT_So (PDB ID code 4UVM) is shown with the conserved TM2 histidine, His61, and extracellular gate residues, Asp316 and Arg32. Asn454 can be seen coordinating the interaction between His61 and Asp316 in this conformation. Sequence logos show the conservation of these residues among the mammalian members of the POT family. (B) Proton-driven uptake of dialanine over time for His61Asp, Asp316His, and the double mutant.

Fig. 4.

Water networks connect proton binding sites within PepT_Xc. (A) Crystal structure of PepT_Xc highlighting the observed extracellular and lateral cavities. Waters are shown as red spheres, bound lipid in yellow, and conserved histidine and aspartate residues in magenta. (B) Cartoon representation of PepT_Xc indicating the waters seen in the crystal structure. (C) Water network observed from the extracellular cavity and the interactions observed within the conserved triad of aspartate, histidine, and asparagine residues. (D) Occupancy profile for water oxygens between Asp322 and Glu425 in PepT_Xc averaged over 100 ns of the Glu425-protonated simulation. Regions where water oxygens exist over 40% of the time are shown in gray and regions with over 60% occupancy are shown in red. (E) Free energy profile (PMF) for proton transfer between Asp322 and Glu425. The reaction coordinates collective variable ${\xi }_R$ transitions from zero when the Asp is protonated to one when the Glu is protonated. The positions of Asp322, Arg37, Lys324, and Glu425 are indicated by text boxes.

Fig. 5.

Protonation of histidine on TM2 initiates inward- to outward-facing transition. Probe radius profiles for the crystal (A) and MD equilibrated structures (B) of PepT_Xc. The constriction along the transporting path is positioned at the extracellular gate in A, implying an inward-open state, while the constriction is positioned at the intracellular gate in B, implying an outward-open state. (C) Close-in view of the extracellular gate showing the conformational change following protonation of His67 from the crystal structure (colored) to MD equilibrated structure (gray). (D) Following protonation of His67, PepT_Xc transitions from inward- to outward-facing conformation. The MD ensembles for His67-protonated (blue), Glu425-protonated (orange), and neither residue protonated (green) are compared with crystal structures of MFS transporters in different conformational states.