Featured Article - April 2015
Short description: In POT transporters, peptide length determines whether the orientation of the ligand is lateral or vertical.
Dipeptides (orange) bind laterally, while tripeptides (purple) adopt a vertical orientation. Both ligands utilize side-chain pockets, but only dipeptides induce formation of a hydrophobic cavity. Figure from ref. 1.
Certain mammalian membrane transporters accept not only nutrients but also molecules, such as drugs. Understanding the promiscuity of ligand binding by these transporters could be of value to drug designers. One such class of transporters is the proton-coupled peptide transporter (POT) family, found in prokaryotic and eukaryotic plasma membranes. POT members have one peptide binding site but can transport >8000 di- or tripeptides, antibiotics, and other drugs.
To understand this site's binding specificity, the Newstead and Caffrey groups (PSI MPID) solved crystal structures of a bacterial POT, PepTS1, in the apo form (PDB 4D2B), with L-Ala-L-Phe dipeptide (PDB 4D2C) and with tri-L-Ala (PDB 4D2D). The dipeptide is positioned laterally via electrostatic interactions with the N- and C-terminal bundles of the transporter. A predicted hydrophobic pocket, not observed in a previous PepTSt structure (PDB 4APS), was found near a Tyr68 residue; the π-π stacking interaction it makes with the dipeptide phenyl ring presumably contributes to specificity. Another pocket seen near the peptide's N-terminus could facilitate binding of larger side chains.
Due to their larger size, tripeptides and antibiotics were unable to have a similar interaction with the binding site; in fact, the tri-Ala peptide structure revealed that the ligand was positioned vertically rather than laterally, although its orientation (C-terminus facing outward or inward) was unresolved. The tripeptide has fewer interactions with the transporter and, assuming the tripeptide's C-terminus faces outward, those occur only in the C-terminus, resulting in a less compact structure than that with the dipeptide. Mutations in the transporter that affect the binding of one or both peptide substrates were identified. The less compact structure of the tripeptide-bound form was confirmed by solving the apo form and comparing all three structures. In the presence of the di- but not the tripeptide, the C-terminal bundle of PepTS1 undergoes a rearrangement to form the hydrophobic pocket, which acts as a gate around the dipeptide.
This analysis suggests that the broader specificity of the POT transporters originates in a binding pocket that is sufficiently plastic (by virtue of its several pockets) to bind ligands in different orientations. Even though ligand binding induces an occluded state of the transporter, this state is different depending on the ligand. It remains possible that even further binding modes may be seen with other ligands.
J. A. Lyons et al. Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters.
EMBO Rep. 15, 886-893 (2014). doi:10.15252/embr.201338403