Fig. 4

Hypothetical model of Asp-Ala bound YePEPT and stabilizing effect of Phe311. a YePEPT structure with a virtually bound Asp-Ala dipeptide (view from the membrane plane): this dipeptide was built by keeping the dipeptide backbone of Ala-Phe (Fig. 3c,f) fixed and mutating the side chains of Ala-Phe into Asp-Ala in Pymol [23]. The rotamer of Asp in Asp-Ala does not introduce any clashes with the YePEPT structure. The distances between the nitrogen atom of Lys314 and the closest oxygen atoms of the carboxyl groups of Asp-Ala (residue at R1 position) and Glu312 are indicated. b The rotamer of Lys314 found in the crystal structure (magenta) and of the rotamer with the shortest distance to the closest oxygen atom of the carboxyl group of Asp-Ala (residue at R1 position) is shown (pale green). c In the YePEPT crystal structure, the interactions between Lys314 and the stabilizing residue Phe311 consist of a hydrogen bond and a pi-cation interaction between the ε-amino group of Lys314, and the carbonyl and phenyl groups of Phe311, respectively. Amino acid residues of YePEPT potentially involved in dipeptide backbone binding are labeled and colored in black (similar to Fig. 3). The N- and C-terminal six-helix bundles are colored in salmon and cyan, respectively. d Mutation of the Lys314 stabilizing Phe311 residue into Ala dramatically reduces the transport function of YePEPT. Expression levels in E. coli of wild-type (wt) and YePEPT^F311A (F311A) used for the uptake experiments were comparable. Error bars in (d) represent SEM from two independent experiments, each in triplicate