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

Abstract

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.