Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter

To elucidate the decisive structural factors relevant for dipeptide–carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly‐Sar transport in Caco‐2 cells. Dipeptides with proline or alanine in the C‐terminal position displayed affinity constants (Ki) of 0.15–1.2 mm and 0.08–9.5 mm, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N‐terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa‐Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24–70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1–0.16 mm. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa‐Pro and Xaa‐Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys‐Pro, Arg‐Pro and Pro‐Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa‐Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa‐piperidides and Xaa‐thiazolidides. Dipeptides with N‐terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro‐Xaa dipeptides such as Pro‐Leu, Pro‐Tyr and Pro‐Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C‐terminal amino acid and the rigidity of the structure.