| The human intestinal dipeptide transporter, hPepT1, has wide substrate specificity making it an ideal target for the absorption of orally delivered drugs. The structure of the substrate translocation pathway of hPepT1 was studied to explain protein-substrate interactions.;The central hypothesis of this study is that there are two categories of amino acids involved in substrate transport: (i) core amino acids are required for substrate transport and are located on transmembrane domains (TMDs) 3, 5, 7, and 10 and (ii) auxiliary amino acids that form substrate interaction pockets are located on TMDs 5, 7, and 10 and are not required for the transport of all substrates. Mapping the locations of these amino acids will aid in explaining hPepT1 substrate specificity and affinity. This hypothesis was tested using computer modeling, mutagenesis, and cysteine scanning mutagenesis.;A computer generated model, termed the convergent model, of hPepT1 was developed to identify TMDs involved in substrate transport. The model suggests that the channel of hPepT1 is created by transmembrane domains 3, 5, 7, and 10. These domains form a football-shaped pore surrounding the second most hydrophic TMD in hPepT1, domain 6. These results suggest that domain 6 might act as a "hydrophobic plug" that prevents substrate transport from occurring without proton binding. The role of TMD 6 was further explored using mutagenesis and methane thiosulfonate (MTS) binding studies. Data indicate that the binding of gly-sar to hPepT1 causes a conformational change. MTSET inhibitions studies indicate that domain 6 does not reside within the substrate channel of hPepT1. Cysteine scanning studies were conducted on TMD 3 to determine if this domain lines the channel of hPepT1. Data clearly indicate that this TMD lies in a tilted position forming part of a funnel for substrate translocation. The exofacial half of the domain may form an accessory binding pocket for larger substrates.;The results obtained from this study elucidate potential roles for TMD 6 in protein conformation during substrate transport and TMD 3 in substrate binding. |
