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Proton movement and coupling in the POT family of peptide transporters

  1. Simon Newsteada,1
  1. aDepartment of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
  2. bDepartment of Chemistry, The University of Chicago, Chicago, IL 60637;
  3. cInstitute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637;
  4. dJames Franck Institute, The University of Chicago, Chicago, IL 60637;
  5. eSchool of Medicine, Trinity College Dublin, Dublin, Ireland;
  6. fSchool of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland
  1. Edited by Christopher Miller, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved November 3, 2017 (received for review June 25, 2017)

  1. Fig. 2.

    PepTSo transports both di- and trialanine using the same number of protons. Steady-state accumulation of di- and trialanine, driven using a fixed ΔμH+, in PepTSt (A) and PepTSo (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.

  2. Fig. 3.

    Conservation of the TM2 histidine in mammalian and mammalian-like POT family transporters. (A) The extracellular cavity from PepTSo (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.

  3. Fig. 4.

    Water networks connect proton binding sites within PepTXc. (A) Crystal structure of PepTXc 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 PepTXc 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 PepTXc 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 <mml:math><mml:mrow><mml:msub><mml:mi>ξ</mml:mi><mml:mi>R</mml:mi></mml:msub></mml:mrow></mml:math>ξR 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.

  4. 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 PepTXc. 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, PepTXc 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.

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