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Peptide transporters yield gating secrets in a new study

A systematic study of a major family of membrane transporters has provided new insights into how they carry out their function.

Structure of the bacterial peptide transporter, PepTSo, in an inward-open conformation, as determined by X-ray crystallography. The two halves of the protein can be seen. (http://bit.ly/pdb-4uvm)

Structure of the bacterial peptide transporter, PepTSo, in an inward-open conformation, as determined by X-ray crystallography. The two halves of the protein can be seen. (http://bit.ly/pdb-4uvm)

The multi-pronged approach, co-ordinated by Dr Philip Fowler in Professor Mark Sansom's lab and Associate Professor Simon Newstead, along with Professor Anthony Watts and others, is published in Structure (1).

By using a diverse range of experimental and computational methods, the team has provided a more complete picture of how the peptide transporters change their shape in order to move molecules across the cell membrane.

The project focuses on two members of this family, PepTSo and PepTSt. These bacterial proton-coupled oligopeptide transporters are homologous to the human transporter PepT1 that is the main route by which the body absorbs dietary protein. PepT1 recognises a diverse range of small peptides and is also responsible for the absorption of many orally administered drugs including most of the beta-lactam antibiotics.

Oligopeptide transporters belong to the much larger Major Facilitator Superfamily (MFS). This includes over 70 different families, members of which share a common mechanism for transporting substrates across the membrane. Molecules bound in the central cavity of the transporter become alternately exposed on either side of the membrane. Access to the site from the other side is prevented by formation of gates - transient constrictions formed by the close packing of several transmembrane helices.

Although experimental structures of many members of the MFS have been determined, all the structures of the peptide transporters have their cytoplasmic gate open ('inward-open' conformation). Details of how the transmembrane helical bundles rearrange around the central ligand-binding site to facilitate transport has therefore been unclear.

The group decided to adopt a systematic approach towards understanding the gating organisation in peptide transporters, and MFS transporters in general. In particular, they wanted to generate plausible models of PepTSo and PepTSt where the cytoplasmic gate was closed and the periplasmic gate was open (the 'outward-open' conformations).

They started with inward-open structures of representative family members, oligopeptide transporters PepTSo and PepTSt, including a high resolution crystal structure of PepTSo recently determined by Professsor Martin Caffrey's lab in Dublin. As their modelling approaches, they took the repeat-swapping method - carried out by collaborator Dr Lucy Forrest and her group, now based at the NIH in Bethesda - and molecular dynamics simulations of the protein embedded in a lipid bilayer.

Using the repeat-swapping method, the researchers were able to generate outward-open models of both proteins. One of the models was then validated by an experimental approach known as DEER spectroscopy, through collaboration with Professor Anthony Watts in the department.

On the left, the structure of PepTSo with the alpha-helices drawn as curved cylinders and coloured according to whether they form the periplasmic (green) or cytoplasmic (blue) gates (or neither, in white). On the right, a model of the same protein is shown in an outward-facing conformation. The model was generated using the repeat-swapping method

On the left, the structure of PepTSo with the alpha-helices drawn as curved cylinders, and coloured according to whether they form the periplasmic (green) or cytoplasmic (blue) gates, or neither (in white). On the right, a model of the same protein is shown in an outward-facing conformation. The model was generated using the repeat-swapping method.

Molecular dynamics simulations of the proteins ran by Dr Fowler provided a second, independent way of predicting what both transporters looked like in the outward-open conformation. For both proteins, the researchers saw changes in their structures indicative of a partial transition towards the outward-open state. Analysis predicted which salt bridges stabilise the outward-open conformation and this was subsequently confirmed experimentally.

By then analysing all the current known structures of MFS proteins, the group was able to identify which transmembrane helices form the periplasmic and cytoplasmic gates - an important detail that had not been investigated before.

Dr Fowler comments that the study has made an important contribution to the field - not only helping to understand how the proton-coupled oligopeptide transporters function but also shedding light on the gating topology of the much wider MFS family.

Reference

  1. Fowler PW, Orwick-Rydmark M, Radestock S, Solcan N, Dijkman P, Lyons JA, Kwok J, Caffrey M, Watts A, Forrest LR and Newstead S. Gating topology of the proton coupled oligopeptide symporters. (2015) Structure 23 290-301

 

 

 

 

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