Figure 1.
The putative peptide binding cavity.
(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.
Figure 2.
Partial sequence alignment of experimentally verified active members (Uniprot accession codes) of the POT family showing the regions of mutations.
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.
Figure 3.
functional analyses of YjdL variants.
(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).
Figure 4.
functional analyses of YdgR variants.
(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).
Table 1.
Primers used for site-directed mutagenesis.
Table 2.
IC50 values of selected dipeptides on YjdL and YdgR variants.
Figure 5.
Inhibition profiles for WT-YjdL (white), Asp392Glu (grey), and Asp392Ser (black).
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).
Figure 6.
Schematic dipeptide orientation model for YjdL exemplified by Tyr-Lys.
The cavity shape has been adapted from PepTSo (Figure 1B). The labeled spheres indicate the position of the C-alpha of the corresponding residue in PepTSo. Residues behind the plane of the peptide are colored brown.
Table 3.
IC50 values of C-terminal lysyl dipeptides.