Tripeptide binding in a proton‐dependent oligopeptide transporter

Abstract

Proton‐dependent oligopeptide transporters (POTs) are important for the uptake of di‐/tripeptides in many organisms and for drug transport in humans. The binding mode of dipeptides has been well described. However, it is still debated how tripeptides are recognized. Here, we show that tripeptides of the sequence Phe‐Ala‐Xxx bind with similar affinities as dipeptides to the POT transporter from Streptococcus thermophilus (PepTS t). We furthermore determined a 2.3‐Å structure of PepTS t in complex with Phe‐Ala‐Gln. The phenylalanine and alanine residues of the peptide adopt the same positions as previously observed for the Phe‐Ala dipeptide, while the glutamine side chain extends into a hitherto uncharacterized pocket. This pocket is adaptable in size and can likely accommodate a wide variety of peptide side chains.

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

“…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 .…”

“…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] .…”

Mechanistic picture for chemo-mechanical couplings in a bacterial proton-coupled oligopeptide transporter from Streptococcus thermophilus

“…17 According to this mechanism, a POT transporter has to undergo at least four major conformational changes for it to facilitate transport, mainly outward-facing (apo), occluded (substrate bound), inward-facing (apo), and occluded (apo). 1,4 All the bacterial POT crystal structures reported to date are either occluded (OCC), 18 inward-facing occluded (IF occ ) or an inward-facing (IF) state; 3,7,[16][17][18]21,22 an outward-facing (OF) state is not yet available. Even the MD studies that were reported earlier on POTs 19,23,24 failed to capture the fully open and stable OF conformation.…”

“…24 PepT St is a bacterial POT symporter from Streptococcus thermophilus. Several studies have been carried to investigate different aspects of PepT St through various experimental 5,18,22,24,27,28 and computational techniques, 24,28,29 including solving protein structures in various conformations, 17,18,22,27 understanding and proposing the functional mechanism, 4,17,24 identifying different substrate binding sites and multiple binding modes for the substrates, 4,5 multispecific substrate recognition pathways, 3,18 mechanism of proton and peptide coupling, 28 different transport mechanisms for different POT transporters, 4,28 and binding of a range of substrates. 29 However, a study utilizing all available conformations of the PepT St is needed to gain a detailed atomistic level understanding of its transport mechanism and the current availability of multiple conformations of PepT St provides a perfect opportunity.…”

“…29 However, a study utilizing all available conformations of the PepT St is needed to gain a detailed atomistic level understanding of its transport mechanism and the current availability of multiple conformations of PepT St provides a perfect opportunity. 17,18,22,27 PepT St is the only POT symporter that has been crystallized in OCC, IF, and IF occ conformational states. 17,18,22,27 Hence, in this study we simulated all three available conformational states of PepT St via unbiased all-atom MD [30][31][32][33] with either E300 protonated or deprotonated.…”