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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)


The uptake of nutrients from the environment is an essential process that is achieved in most cells through the use of secondary active transporters. The POT family of proton-coupled peptide transporters are one of the most diverse nutrient uptake systems, recognizing amino acids, peptides, nitrate, and seed-defense compounds. A long-standing question is how this family achieves such ligand diversity. A high-resolution crystal structure combined with multiscale molecular dynamics simulations demonstrate water molecules are able to shuttle protons using a Grotthuss-type mechanism, suggesting a separation of ligand recognition from proton movement. This would have clear advantages for a transporter family that must accommodate chemically diverse ligands while retaining the ability to couple transport to the proton electrochemical gradient.


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.


  • ?1To whom correspondence may be addressed. Email: joanne.parker{at}bioch.ox.ac.uk, jmswanson{at}uchicago.edu, or simon.newstead{at}bioch.ox.ac.uk.
  • Author contributions: J.L.P., J.M.J.S., M.C., G.A.V., and S.N. designed research; J.L.P., C.L., A.B., Z.W., L.V., N.S., G.L.-V., J.M.J.S., and S.N. performed research; J.L.P., C.L., and M.C. contributed new reagents/analytic tools; J.L.P., C.L., J.M.J.S., M.C., G.A.V., and S.N. analyzed data; and J.L.P., C.L., J.M.J.S., and S.N. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 6EI3).

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1710727114/-/DCSupplemental.

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