Structural and functional studies of nutrient and drug uptake systems

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.

List of scientific papers

I. High-throughput analytical gel filtration screening of integral membrane proteins for structural studies. Löw C, Moberg P, Quistgaard EM, Hedrén M, Guettou F, Frauenfeld J, Haneskog L and Nordlund P. Biochim. Biophys. Acta. 1830(6), 3497-508 (2013).
https://doi.org/10.1016/j.bbagen.2013.02.001

II. Structural insights into substrate recognition in proton-dependent oligopeptide transporters. Guettou F, Quistgaard EM, Trésaugues L, Moberg P, Jegerschöld C, Zhu L, Jong AJ, Nordlund P and Löw C. EMBO Rep. 14(9), 804-10 (2013).
https://doi.org/10.1038/embor.2013.107

III. Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2. Guettou F, Quistgaard EM, Raba M, Moberg P, Löw C and Nordlund P. Nat. Struct. Mol. Biol. 21(8), 728-31 (2014).
https://doi.org/10.1038/nsmb.2860