Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter

Abstract

SummaryProton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug absorption. These transporters accept a wide range of substrates, but the structural basis for how different peptide side chains are accommodated has so far remained obscure. Twenty-eight peptides were screened for binding to PepTSt from Streptococcus thermophilus, and structures were determined of PepTSt in complex with four physicochemically diverse dipeptides, which bind with millimolar affinity: Ala-Leu, Phe-Ala, Ala-Gln, and Asp-Glu. The structures show that PepTSt can adapt to different peptide side chains through movement of binding site residues and water molecules, and that a good fit can be further aided by adjustment of the position of the peptide itself. Finally, structures were also determined in complex with adventitiously bound HEPES, polyethylene glycol, and phosphate molecules, which further underline the adaptability of the binding site.

Tripeptide binding in a proton‐dependent oligopeptide transporter

“…We conclude that both the DSF and MST results indicate that bulky apolar residues are preferred over smaller polar/charged ones in the third position of the Phe‐Ala‐Xxx‐ peptides. It may also be noted that the affinity of Phe‐Ala‐Leu is higher than for most of the previously tested dipeptides, and only 2.1‐fold lower than for the best binding one, Ala‐Leu, which displayed an affinity of 0.56 ± 0.08 m m . Thus, while it is possible that a systematic analysis of all di‐ and tripeptides would bear out the notion that PepT St on average prefers dipeptides, it is evident that some tripeptides also bind quite well.…”

“…The Phe‐Ala‐Phe tripeptide was only soluble in DMSO (100 m m stock), which did not allow us to determine a full binding isotherm using MST. As the Phe‐Ala dipeptide displays a K D value of 10.95 ± 2.2 m m , we can infer that: (a) Extending this peptide with an extra alanine residue has no effect on the binding affinity, indicating that it can be accommodated in the binding site, but without strengthening the interaction. (b) Adding instead a threonine or aspartate residue moderately or strongly reduces affinity, respectively, suggesting that especially the latter is clashing within the binding site.…”

“…The strongest effect was observed for peptides with a bulky apolar residue in the third position, that is, Phe‐Ala‐Leu and Phe‐Ala‐Phe. These two peptides were also more stabilizing than any of the previously tested tripeptides, although markedly less compared to the best performing dipeptides . Next, we used MST to further characterize the binding of Phe‐Ala‐Xxx peptides to PepT St (Fig.…”

“…S1D–F). However, in a recent effort to characterize binding of different dipeptides to PepT St , we obtained data suggesting that the observed vertically bound molecule in PepT St might instead have been a misidentified HEPES buffer molecule . There is, therefore, an outstanding need for more structural insights into how tripeptides are recognized by POTs.…”

“…During the transport cycle, these domains move relative to each other to allow alternate access from the cytoplasmic and extracellular sides . Several X‐ray structures of bacterial POTs have been reported, either in the apo‐form , peptide bound state , or in complex with the phosphonodipeptide alafosfalin (Table S1). However, only three structures have hitherto been reported in which a tripeptide is bound.…”