Primers used for site-directed mutagenesis.

<p>IC₅₀ values (mM) with SEM (n ≥3) of selected dipeptides tested on YjdL and YdgR variants.</p>a<p>IC₅₀ values determined at 0.2 mM β-Ala-Lys(AMCA) and 5 min incubation.</p>b<p>IC₅₀ values determined at 0.5 mM β-Ala-Lys(AMCA) and 15 min incubation.</p

Schematic dipeptide orientation model for YjdL exemplified by Tyr-Lys.

<p>The cavity shape has been adapted from PepT_So (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047780#pone-0047780-g001" target="_blank">Figure 1B</a>). The labeled spheres indicate the position of the C-alpha of the corresponding residue in PepT_So. Residues behind the plane of the peptide are colored brown.</p

IC₅₀ values of C-terminal lysyl dipeptides.

<p>Forward primer sequences used to generate mutations in <i>yjdL</i> by site-directed mutagenesis. The reverse complement of the forward primers was used as the reverse primers.</p

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from <em>E. coli</em>

<div><p>YjdL from <em>E. coli</em> is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical <em>E. coli</em> POT, were compared in light of the crystal structure of a POT from <em>Shewanella oneidensis</em>. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.</p> </div

functional analyses of YdgR variants.

<p>(A) Representative western blots of YdgR mutants. (B) β-Ala-Lys(AMCA) uptake (0.2 mM, 5 min) by YdgR mutants in uptake buffer, pH 6.5. Error bars indicate SEM (n ≥3). Lys133Arg and Lys133Gln are not significantly different, P>0.05, from background levels (pTTQ18).</p

IC₅₀ values of selected dipeptides on YjdL and YdgR variants.

<p>IC₅₀ values (mM) with SEM (n ≥3) of C-terminal lysine dipeptides tested on YjdL variants.</p>a<p>IC₅₀ values determined at 0.2 mM β-Ala-Lys(AMCA) and 5 min incubation.</p>b<p>IC₅₀ values determined at 0.5 mM β-Ala-Lys(AMCA) and 15 min incubation.</p

Inhibition profiles for WT-YjdL (white), Asp392Glu (grey), and Asp392Ser (black).

<p>The cells were incubated in assay buffer pH 6.5 containing 0.5 mM β-Ala-Lys(AMCA) and 50 mM ligand. Error bars indicate SEM (n ≥3).</p

functional analyses of YjdL variants.

<p>(A) Representative western blots of YjdL mutants. (B) β-Ala-Lys(AMCA) uptake (0.2 mM, 5 min) by YjdL mutants in uptake buffer, pH 6.5. Error bars indicate SEM (n ≥3). Tyr25Ala, Tyr58Ala, and Tyr113Ala are not significantly different, P<0.05, from background levels (pTTQ18).</p

The putative peptide binding cavity.

<p>(A) View of the PepTSo structure (PDB ID: 2XUT). The N-terminal and C-terminal domains are colored blue and red, respectively, and amino acids constituting the putative peptide binding site are represented as sticks. Helix VII and VIII are semi transparent for better overview of the putative binding site. (B) View of the putative peptide binding cavity of PepTSo. Residues mutated in this study have been colored brown. The cavity shape is shown as a thin slab centered on atoms Ser423 OG, Lys127 NZ, and Glu419 OE2. TDG from the lactose permease structure (PDB ID: 1PV7) is shown as transparent spheres. The dotted line marks the division of the cavity into two pockets.</p

Partial sequence alignment of experimentally verified active members (Uniprot accession codes) of the POT family showing the regions of mutations.

<p>The numbering corresponds to YjdL residues. Sequences boxed in black belong to bacterial and eukaryotic POTs, while those boxed in green belong to plant POTs. Residues deviating from the POT consensus are highlighted by a red background.</p