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 (K i ) 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 K i values for the incompetent cis isomers, 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 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 K i 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.

A Quantitative Structure–Activity Relationship for Translocation of Tripeptides via the Human Proton-Coupled Peptide Transporter, hPEPT1 (SLC15A1)

“…molecules translocated). Brandsch et al investigated the effect of Pro position in dipeptides and found that X aa -Pro dipeptides generally have higher affinity than Pro-X aa dipeptides (28), which corresponds well with our findings.…”

“…All experiments were conducted three days postseeding. MDCK/hPEPT1 cells were used in passages [25][26][27][28][29][30][31][32][33][34][35], and the MDCK/Mock cells were in passage 20-31.…”

“…The cyclic nature of Pro limits the flexibility of Procontaining peptides (28), which could affect the substrate translocation via hPEPT1. Two of the tripeptides with Pro in the middle position (Arg-Pro-Ser and Trp-Pro-Tyr) were translocated whereas two others were not (Met-Pro-Pro and Gly-Pro-Lys).…”

“…hPEPT1 is assumed only to translocate dipeptides in their trans-conformation (28), and the presence of Pro in a dipeptide shifts the cis-trans equilibrium in the direction of increased cis-conformation. This might also explain why Pro containing tripeptides are poor transportates (i.e.…”

An update on renal peptide transporters

“…Large sets of dipeptides, amino acid derivatives, or peptidomimetics have been probed for transport in competition experiments utilizing cells expressing either one of the transporters, by the measurement of substrate-mediated transport currents in Xenopus laevis oocytes expressing PEPT1 or PEPT2, or by competition experiments in the yeast Pichia pastoris heterologously expressing either one of the mammalian transporters (11,12,14,21,22,66,67). One important finding for understanding the "multispecificity" of the peptide transporters was that neither PEPT1 nor PEPT2 requires a peptide bond in a substrate and that the minimal structural requirement in a substrate for binding and transport is a simple carbon chain that separates the oppositely charged NH 2 and COOH head groups by an intramolecular distance of Ͼ 500 Ͻ 630 picometers (21).…”