Abstract
Orally ingested nutrients and drugs are selectively absorbed from our small intestines into the
bloodstream through various membrane-integrated transporters. The present study focuses
mainly on a specific absorption route, namely the proton dependent oligopeptide transporters
(POTs). These transporter systems belong to the major facilitator superfamily (MFS) and are
secondary active transporters. The aim of this thesis was to study the structure and
mechanism of POTs from prokaryotic organisms. The project was divided in two phases.
During the first phase, a high-throughput method was developed for rapid screening of
integral membrane proteins (IMP) to identify suitable targets, constructs, and production
conditions for structural studies (paper I). During the second phase of the project, X-ray
crystal structures of the prokaryotic peptide transporter (PepTSo2) from the organism
Shewanella oneidensis in complex with four different substrates were determined (paper IIIII).
The structures revealed the overall conformational state of the protein as well as the
architecture of the substrate-binding site. The protein was captured in an inward open
conformation where the substrate-binding site was accessible to the cytoplasm but not to the
periplasm. The bound peptides adopted extended lateral conformations with their N-termini
interacting with a conserved polar pocket while their C-termini were in close proximity to a
positively charged pocket. The results presented in papers I-III provide novel structural and
mechanistic insights into prokaryotic peptide transporters. Interestingly, the binding site
residues are highly conserved in the human peptide transporter homolog, PepT1. Hence,
these results not only increase our understanding regarding prokaryotic peptide transporters
but also shed light on the human homologs. Furthermore, results presented in this work may
assist in design of pharmacologically active compounds into substrates of the human peptide
transporter, creating orally administrated drugs.
