Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2

Abstract

Peptide transporters of the PepT family have key roles in the transport of di- and tripeptides across membranes as well as in the absorption of orally administered drugs in the small intestine. We have determined structures of a PepT transporter from Shewanella oneidensis (PepT(So2)) in complex with three different peptides. The peptides bind in a large cavity lined by residues that are highly conserved in human PepT1 and PepT2. The bound peptides adopt extended conformations with their N termini clamped into a conserved polar pocket. A positively charged patch allows differential interactions with the C-terminal carboxylates of di- and tripeptides. Here we identify three pockets for peptide side chain interactions, and our binding studies define differential roles of these pockets for the recognition of different subtypes of peptide side chains.

Peptide Selectivity of the Proton-Coupled Oligopeptide Transporter from <b><i>Neisseria meningitidis</i></b>

“…On the contrary YdgR and YhiP from E. coli show specificity to a much lesser extent [Harder et al, 2008;Weitz et al, 2007]. Other [Guettou et al, 2014] and YePEPT from Yersinia enterocolitica [Boggavarapu et al, 2015], which both have a preference for an acidic residue on the N-terminal residue of a dipeptide, and in the case of PepT So also in a tripeptide [Prabhala et al, 2014a]. For comparison with NmPOT, a more relevant example is DtpT from Lactococcus lactis ; in the case of this transporter the apparent affinity for dipeptides with a positively charged side chain in the N-terminal position decreases 80-fold compared to Ala-Ala, but no change is observed when a positively charged side chain is introduced in the N-terminus.…”

“…In recent years several POT crystal structures have been determined [Doki et al, 2013;Guettou et al, 2014;Lyons et al, 2014;Newstead et al, 2011]. They all contain 14 transmembrane helices, arranged in two 6-helix bun- dles connected by a region consisting of two additional transmembrane helices [Lyons et al, 2014].…”

“…All structures determined so far adopt an inward facing conformation, which exposes the binding site to the cytoplasm and may thus represent substrate release. Given the fact that the transporters of this family are able to transport peptides in the reverse direction as well [Jensen et al, 2014], valuable knowledge of the peptide-binding determinants has been inferred from the POT structures crystallized in a complex with peptides and complementary mutational studies [Doki et al, 2013;Guettou et al, 2014;Jensen et al, 2014;Lyons et al, 2014]. To investigate the structural basis of the selectivity shown by NmPOT, we created a 3D homology model of NmPOT based on the structure of GkPOT (PDB 4IKV [Doki et al, 2013]) and placed the dipeptide Ala-Ala from its complex with the POT PepT So2 from S. oneidensis (PDB 4TPH [Guettou et al, 2014]) as a template to study dipeptide:POT interactions.…”

Symmetry and Structure in the POT Family of Proton Coupled Peptide Transporters

“…Insights into the biochemical basis for peptide binding and selectivity have recently come from three different POT family members crystallised in complex with peptides [17,34,35]. Detailed analyses of the binding interactions in these structures have been published recently [19,29] and will not be discussed in detail here.…”

“…These two regions are opposite an acidic patch on TM7 and 10 in PepT St and a hydrophobic pocket, constructed of aromatic side chains from TM's 2, 11. Several lines of biochemical evidence suggest that ligand selectivity is the result of interactions between the side chains of the peptide and these pockets [34][35][36], which in turn promote the conformational changes required for transport. It is unclear what role, if any, symmetry plays in peptide recognition.…”