MINIMAL MOLECULAR DETERMINANTS OF SUBSTRATES FOR RECOGNITION BY THE INTESTINAL PEPTIDE TRANSPORTER

Proton-dependent electrogenic transporters for di- and tripeptides hav e been identified in bacteria, fungi, plants, and mammalian cells. The y all show sequence-independent transport of all possible di- and trip eptides as well as of a variety of peptidomimetics, We used the mammal ian intestinal peptide transporter PEPT1 as a model to define the mole cular basis for its multisubstrate specificity. By employing computati onal analysis of possible substrate conformations in combination with transport assays using transgenic yeast cells and Xenopus laevis oocyt es expressing PEPT1, the minimal structural requirements for substrate binding and transport were determined. Based on a series of medium ch ain fatty acids bearing an amino group as a head group (omega-amino fa tty acids, omega-AFA), we show that electrogenic transport by PEPT1 re quires as a minimum the two ionized head groups separated by at least four methylene groups. Consequently, a > 500 pm < 630 pm distance betw een the two charged centers (carboxylic carbon and amino nitrogen) is sufficient for substrate recognition and transport. Removal of either the amino group or the carboxyl group in omega-AFA maintained the affi nity of the compound for interaction with the transporter but abolishe d the capability for electrogenic transport, Additional groups in the omega-AFA backbone that provide more hydrogen bonding sites appear to increase substrate affinity but are not essential. The information pro vided here does (a) explain the capability of the peptide carrier for sequence-independent transport of thousands of different substrates an d (b) set the molecular basis for a rational drug design to increase t he absorption of peptide-based drugs mediated by PEPT1.