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 int estinal H+/peptide symporter (PEPT1) was investigated by measuring their ab ility to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (K-i) o f 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationshi p 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 b etween the cis/trans ratios (trans contents 24-70%) and the affinity consta nts was observed. After correcting the K-i values for the incompetent cis i somers, the K-i 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 conformati on even for a transport protein that is quite promiscuous in substrate tran slocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipepti des, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, A rg-Pro and Pro-pro indicate that additional molecular factors affect their binding at PEPT1. The K-i values obtained for the corresponding Xaa-Ala dip eptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xa a-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline dis played a very diverse affinity profile. However, in contrast to current kno wledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPTI with appreciable affinities. Binding seems mainly dete rmined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.