Structure of Prototypic Peptide Transporter DtpA fromE. coliin Complex with Valganciclovir Provides Insights into Drug Binding of Human PepT1

Abstract

Members of the solute carrier 15 family (SLC15) transport di- and tripeptides as well as peptidomimetic drugs across the cell membrane. Structures of bacterial homologues have provided valuable information on the binding and transport of their natural substrates, but many do not transport medically relevant drugs. In contrast, a homologue from Escherichia coli, DtpA (dipeptide and tripeptide permease), shows a high similarity to human PepT1 (SLC15A1) in terms of ligand selectivity and transports a similar set of drugs. Here, we present the crystal structure of DtpA in ligand-free form (at 3.30 Å resolution) and in complex with the antiviral prodrug valganciclovir (at 2.65 Å resolution) supported by biochemical data. We show that valganciclovir unexpectedly binds with the ganciclovir moiety mimicking the N-terminal residue of a canonical peptide substrate. On the basis of a homology model we argue that this binding mode also applies to the human PepT1 transporter. Our results provide new insights into the binding mode of prodrugs and will assist the rational design of drugs with improved absorption rates.

The conformational distribution of a major facilitator superfamily peptide transporter is modulated by the membrane composition

“…S14). We therefore conclude that the inward-open conformer of DtpA is very stable in LMNG and cannot easily be shifted to alternative conformations, a finding that is in accord with previous x-ray structures that only spotted marginal changes in DtpA in the prodrug bound and apo-state 57 .…”

“…POTs have been extensively studied by x-ray crystallography and biochemical transport assays over the past years 27,36,39,59,70,73,83,84 . Numerous structures of various bacterial homologues in the absence and presence of substrates, drugs, and prodrugs are available, highlighting crucial residues for substrate binding and proton coupling [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] . Unfortunately, only inward-open and partially inward-open states of POTs have so far been described at atomic resolution and the influence of the membrane environment on structure and transport has been neglected 41,42,47,52,53,55,58 .…”

“…s18). Although the prodrug bound x-ray structure also does not show substantial structural changes compared to the apo-structure 57 , the invariance of the conformational distribution of DtpA to substrate in SapNPs is surprising. However, in cells, substrate concentrations differ significantly between periplasm and cytoplasm, thus generating an asymmetry that is not reproduced in our experiments.…”

“…DtpA variants were expressed and purified as previously described 57 . The constructs were transformed into E. coli strain C41(DE3) and grown in TB medium supplemented with 30 µg/ml kanamycin at 37°C.…”

“…Their remarkable substrate promiscuity 38,39 together with their highly abundant MFS fold 40 makes them ideal models to understand the interplay between conformational changes and transport. Numerous bacterial POT structures have been determined to date [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] . We focus on the E. coli peptide transporter DtpA 59,60 to explore the effect of different environments such as detergents and Saposin nanoparticles 61,62 (SapNPs) with different lipid compositions on the conformation of the protein using single molecule FRET (smFRET) [63][64][65][66][67][68] .…”