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Abstract

The uptake of peptides plays a crucial role in metabolism and inflammation. In mammals, peptide absorption and reabsorption are mediated by the proton coupled oligopeptide transporters (POTs) PepT1(SLC15A1) and PepT2 (SLC15A2), of the solute carrier family 15. POTs are one the most promiscuous transporters among solute carriers and constitute the main route of entry for orally administrated peptidomimetic drugs. SLC15 transporters are involved in various inflammatory diseases, and the paralogue PHT1 (SLC15A4) was recently identified as therapeutic target in Systemic Lupus Erythematosus (SLE). The three dimensional structures of several bacterial homologues have been determined in the past 10 years, but how these shuttle systems adapt to such an array of substrate remains poorly understood on the molecular level. In addition, these past snapshots represented exclusively ‘inward facing’ conformations, therefore limiting our molecular understanding of the transitions required to complete an entire transport cycle. In a first step, we determined high resolution three dimensional structures of the prototypical POT DtpB from E. coli, bound to 14 different di- and tripeptides, using macromolecular crystallography (MX). This work provides a profound basis for understanding promiscuity and ligand recognition in POTs at the molecular level. Second, I used single particle analysis cryogenic electron microscopy (SPA cryo-EM), to determine the first structures of the human peptide transporters 1 (HsPepT1) and 2 (HsPepT2). Human PepT1 and PepT2 were captured in four different states throughout the transport cycle, providing a dynamic molecular understanding of substrate uptake within the SLC15 family. Third, we continued using SPA cryo-EM to determine the first structure of PHT1, in an outward facing conformation. This work provides a framework to determine the structure of this newly identified target of SLE, which could be used to obtain high resolution data with various therapeutics. Last, I determined the first structure of the atypical POT DtpC, from E. coli. In this work, we explored various fiducial strategies, to improve the resolution of the reconstruction of MFS transporters in general, and provided a molecular explanation for the selectivity of DtpC towards positively charged dipeptides. In summary, this work delivers new insights into the working principles of proton coupled oligopeptide transporters, and will serve as a reference for future structure based drug design (SBDD) studies targeting members of this family.