Structural and functional analyses of the Escherichia coli peptide transporter DtpA

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dc.contributor.advisor Wagner, Samuel (Prof. Dr.) Ural-Blimke, Yonca 2019-02-14T11:29:42Z 2019-02-14T11:29:42Z 2019-02-14
dc.identifier.other 517715511 de_DE
dc.identifier.uri de_DE
dc.description.abstract The proton-dependent oligopeptide transporters (POTs) are ubiquitous in all kingdoms of life and the only peptide transport system in humans. Coupled to co- transport of protons, they take up di- and tripeptides into the cell. Two human homologues are crucial for the nutrient (re)absorption and drug delivery. Human PepT1 (hPepT1, SLC15A1) is highly expressed in the small intestines, whereas hPepT2 (SLC15A2) is mainly found in the kidney. Indeed, antibiotics such as ceftibuten, medication against high blood pressure such as enalapril and antiviral drugs such as valacyclovir and valganciclovir are taken up by hPepT1 and hPepT2. Although these transporters have been studied biochemically in the past, structural information on any of the eukaryotic transporters is missing. High-resolution structures from bacterial homologues contributed to our understanding of ligand binding and selectivity but these transporters do not transport drugs. Exceptionally, a homologue from Escherichia coli, DtpA, shows a striking similarity to hPepT1 in terms of ligand selectivity and transports several different drug molecules. To characterize the ligand selectivity of DtpA further, I used a number of in vitro and in vivo methods: A ligand library consisting of di- and tripeptides and selected drugs was screened by differential scanning fluorimetry and microscale thermophoresis. Further, the screening was complemented by the established in vivo transport and competition assay. Both in vivo and in vitro data were in good agreement with the following conclusions: (1) DtpA has a significantly higher preference for tripeptides than dipeptides, and (2) prefers di- and tripeptides with hydrophobic and aromatic residues compared to charged residues. (3) Glycine containing ligands are less likely to bind and be transported by DtpA. From the drug library, valacyclovir and valganciclovir stood out with binding affinities similar to the ones the measured for di- and tripeptides. The structure of DtpA was determined using a nanobody as a crystallization chaperone in a ligand-free state at 3.3 Å and a valganciclovir-bound state at 2.7 Å. Valganciclovir, L-valyl ester of ganciclovir, is an antiviral prodrug used in the treatment of cytomegalovirus. The valine moiety increases the solubility and absorption rate of the compound. In fact, ganciclovir without the N-terminal valine cannot be transported by hPepT1 or hPepT2. Because the N-terminal residue of a di-/tripeptide plays an important role for the ligand coordination in previous ligand- bound bacterial structures, the valine moiety was suggested to be coordinated in the position of the N-terminal residue of a di-/tripeptide. However, in the DtpA- valganciclovir structure, the guanine ring mimics the N-terminal residue and the valine moiety is coordinated in a previously uncharacterized pocket. This pocket is larger in DtpA compared to other studied POTs due to a structural difference in transmembrane helix 10 caused by an intrahelical loop. Transmembrane helix 11 is therefore shifted away from the ligand-binding site, which increases the pocket size and allows the transport of various drug molecules. In summary, I present the functional characterization of DtpA by combining in vivo and in vitro studies with a focus on ligand selectivity and bring new insights to this transporter. Furthermore, the DtpA-valganciclovir structure reveals an unexpected binding mode of the drug and an uncharacterized pocket within the ligand-binding site. Finally, we provide a homology model of hPepT1 laying the groundwork for further drug development. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podok de_DE
dc.rights.uri de_DE
dc.rights.uri en
dc.subject.classification Biochemie , Strukturbiologie de_DE
dc.subject.ddc 500 de_DE
dc.subject.ddc 540 de_DE
dc.subject.ddc 570 de_DE
dc.title Structural and functional analyses of the Escherichia coli peptide transporter DtpA en
dc.type PhDThesis de_DE
dcterms.dateAccepted 2018-11-27
utue.publikation.fachbereich Biochemie de_DE
utue.publikation.fakultaet 7 Mathematisch-Naturwissenschaftliche Fakultät de_DE


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