Figure 3

Model of the substrate binding site of YePEPT. (a) YePEPT^WT structure (PDB ID code: 4W6V³²) with modelled bound Asp-Ala dipeptide. (b) YePEPT^K314A-F311Y structure with modelled bound Asp-Ala dipeptide. Distances between the nitrogen atom of K314 and the carboxyl group of the aspartate side chain of Asp-Ala (a), between the hydroxyl group of Y311 and the α-amino group of Asp-Ala (b), and between the carboxyl group of E420 and the α-amino group of Asp-Ala (b) are indicated. Conserved residues involved in peptide backbone interactions in POTs are coloured in black^34,35. Non-conserved residues in the substrate binding pocket are coloured in purple. The N- and C-terminal bundles of the YePEPT^WT structure are coloured in green and blue, respectively. The Asp-Ala dipeptide was modelled into the binding pocket of YePEPT^WT (PDB ID code: 4W6V) by superposition with the Ala-Phe-bound structure of PepT_st in PyMol (The PyMol Molecular Graphics System, Schrödinger), followed by mutagenesis of the dipeptide as described³².