Peptide transport: two transporters for the price of one
Two new studies reveal how a family of peptide transporters is able to transport thousands of different peptides into cells.
Associate Professor Simon Newstead and his group, along with collaborators at Trinity College Dublin and NIH Bethesda, show that the bacterial transporter PepT1 uses different coupling mechanisms to accommodate and transport a diverse range of peptides.
Lipid Cubic Phase crystallisation of bacterial POT family transporters has revealed key insights into the peptide binding site. Shown here the Ala-Phe complex, which reveals the horizontal binding mode (PDB: 4D2C) (Click to enlarge)
The results, in EMBO Reports (1) and eLife (2), suggest that if the human peptide transporter also uses two mechanisms, then companies could hone their strategy for targeting transporters - which also take up antibiotics and antivirals - for drug uptake.
A key characteristic of the proton-dependent oligopeptide transporters (POTs) is their ability to recognise over 8000 different combinations of di- and tri-peptides. The family includes mammalian members PepT1 and PepT2 which are the major route for dietary peptide absorption in the small intestines and kidneys and which also actively take up a number of clinically important drugs. The molecular features of the POT family that give rise to such promiscuity are unknown.
Now the work of Simon Newstead's group and colleagues reveals the tricks that the transporters use to accomplish this.
They approached the problem in two different ways - using high-resolution structural studies and thermodynamics.
In collaboration with Martin Caffrey at Trinity College Dublin, the group used cubic phase crystals to explore for the first time the high-resolution atomic structures of different peptides bound in the transporter.
The results suggested that the di- and tri-peptides might occupy distinct binding sites in the transporter - one more horizontal, the other more vertical.
Professor Newstead comments that the finding was unexpected. 'What we observed was reminiscent of multidrug exporters where there are different sites for different classes of drugs, but it was unexpected for nutrient importers.'
The possibility of there being two distinct sites in the transporter could explain the promiscuity of the transporter. But the group wanted to know whether there were also two distinct mechanisms of transport. 'This would be a novel idea as we would expect the peptides to use the same mechanism,' he adds.
To tackle this question, Dr Jo Parker in the group devised a way of looking at the thermodynamics of the uptake process. For the study, they collaborated with Joseph Mindell at NIH Bethesda and used a novel transport assay that tracks the number of protons per peptide taken up in reconstituted liposomes.
The studies demonstrate a new two mechanism model for proton:peptide symport in PepTSt. For the same proton gradient across the membrane, di-peptides will be concentrated at >1000 more than tri-peptides (Click to enlarge)
The results, described in the eLife paper, show that di-peptides and tri-peptides are transported by a different number of protons. Whilst tri-peptides are transported with a proton:peptide stoichiometry of 3:1, di-peptides are co-transported with either 5 or 6 protons.
Professor Newstead comments that this is the first study to reveal multiple stoichiometries for different ligands through the same transporter. 'It shows that the transporter has evolved different coupling mechanisms to accommodate and transport chemically and physically diverse ligands across the membrane. It's unprecedented to have two mechanisms operating within the same transporter.'
He adds that if mammalian transporters show similar stoichiometry, this has important implications for the development of drugs to use the transporters.
'It really changes how drug companies should be targeting transporters for drug uptake. We should be targeting the di-peptide mechanism because this is a more efficient way of uptake. With a more efficient way of concentrating the drug, lower amounts of drug would need to be administered.'
'To target this mechanism, we need to carry out a more in-depth analysis to understand how di- versus tri-peptides bind.'
It is possible that the two-mechanism feature could operate in other classes of promiscuous transporters, for example the OATPs (Organic Anion-Transporting Polypeptide) and OATs (Organic Anion Transporters) - transporters that drug companies are also trying to target for drug uptake.