Peptide transporter structure reveals binding and action mechanism of a potent PEPT1 and PEPT2 inhibitor

Abstract

Inhibitors for membrane transporters have been shown to be indispensable as drugs and tool compounds. The proton-dependent oligopeptide transporters PEPT1 and PEPT2 from the SLC15 family play important roles in human and mammalian physiology. With Lys[Z(NO₂)]-Val (LZNV), a modified Lys-Val dipeptide, a potent transport inhibitor for PEPT1 and PEPT2 is available. Here we present the crystal structure of the peptide transporter YePEPT in complex with LZNV. The structure revealed the molecular interactions for inhibitor binding and a previously undescribed mostly hydrophobic pocket, the PZ pocket, involved in interaction with LZNV. Comparison with a here determined ligand-free structure of the transporter unveiled that the initially absent PZ pocket emerges through conformational changes upon inhibitor binding. The provided biochemical and structural information constitutes an important framework for the mechanistic understanding of inhibitor binding and action in proton-dependent oligopeptide transporters.

Fig. 1. Influence of LZNV on the thermostability and function of YePEPT.

a Thermal shift assay using YePEPT^WT and YePEPT^K314A, and LZNV. The T_i-values of YePEPT^WT and YePEPT^K314A were determined at different concentrations of LZNV (200 µM, 400 µM and 800 µM). ΔT_i-values at specified LZNV concentrations were calculated by subtraction of the T_i-value in the absence of LZNV. Data points represent mean ± SD of at least three independent experiments. b Kinetics of [³H]Ala-Ala uptake in E. coli cells transformed with the YePEPT^K314A construct. Data points represent uptake of [³H]Ala-Ala normalised to V_max ± SEM of three independent experiments, each at least in triplicate. c IC₅₀ determination of YePEPT^K314A for LZNV by heterologous competition. Data points represent uptake of [³H]Ala-Ala normalised to the uninhibited signal ± SEM of three independent experiments, each at least in triplicates. Numbers in brackets below K_m- and IC₅₀-values represent 95% confidence intervals. If not visible, error bars are smaller than symbols.

Fig. 2. Electron density and overall structure of YePEPT^K314A with bound LZNV.

a Electron density map of the YePEPT^K314A crystal structure. Displayed is the 2Fo-Fc electron density map of a structural fragment of TM7 contoured at 1.0 σ and coloured in blue, and the YePEPT^K314A structure as yellow sticks. The shown fragment of TM7 corresponds to the sequence 293-RLLVCFILLVSAAFFWSAFEQ-313, whereby residues in bold are labelled in the panel. b and c Overall structure of YePEPT^K314A with bound LZNV in an inward-facing conformation. The structure is viewed as from the membrane plane (b) and from the periplasm (c). Grey horizontal lines in b indicate the orientation of YePEPT^K314A in the biological membrane according to the PPM-server. In b and c, the bound LZNV molecule is shown in purple as space-filling model. The N- and C-termini are indicated in capital letters and all 14 TMs are labelled N-terminally (b) or at the periplasmic side (c). The pseudo-twofold symmetrical N- and C-terminal six-helix bundles are coloured in blue and yellow, respectively, whereas the two bundles connecting helices (Ha and Hb) are depicted in black.

Fig. 3. LZNV binding pocket in YePEPT^K314A and molecular protein-ligand interactions.

a Chemical structure of LZNV. Polar and ionic groups are labelled and highlighted in red. b Overall structure of YePEPT^K314A with bound LZNV oriented in the lipid membrane according to the PPM-server. The location of the binding pocket in the transporter is indicated by a black box. c Detailed view into the LZNV binding pocket. Polar and ionic interactions of LZNV (black sticks) with amino acid residues (yellow sticks) located in the binding pocket of YePEPT^K314A are displayed. All polar and ionic interactions within a distance ≤3.5 Å from amino acid residues to the ligand are shown as dashed lines (distances in Å). Amino acids are labelled in the one-letter code and corresponding TMs are indicated in brackets. For N344, the interaction with the main-chain nitrogen is indicated by N344(N).

Fig. 4. LZNV binding pockets of YePEPT^K314A.

a Accommodation of substrate/ligand side chains in the previously described pockets P1 (purple) and P2 (green), and of the Z(NO₂) moiety in the newly identified pocket PZ (yellow). b Detailed view into the PZ pocket, accommodating the Z(NO₂) moiety of LZNV. The π-stacking interaction between F318 and the 4-nitrophenyl group of LZNV is displayed with two balls located in the centre of the aromatic rings and connected by a dashed line (cyan). The position of K314 in the structure of YePEPT^WT is shown in red. Protein side chains involved in pocket formation are depicted as thin (a) or thicker (b) sticks and colour-coded according to corresponding pocket colour. Amino acids are labelled in the one-letter code and corresponding TMs are indicated in brackets.

Fig. 5. Thermal stabilisation of different YePEPT variants by LZNV.

The T_i-values of different YePEPT variants were determined at three different concentrations of LZNV, i.e., 200 µM, 400 µM and 800 µM. ΔT_i-values at the specified LZNV concentrations were determined by subtraction of the T_i-value in the absence of LZNV. Data points represent mean ± SD of at least three independent experiments.

Fig. 6. Conformational changes of YePEPT^K314A and formation of the PZ pocket upon binding of LZNV.

a Structural alignment of overall structures of the apo (blue ribbon) and the LZNV bound YePEPT^K314A (red ribbon). The location of largest displacement between the apo and LZNV bound structures is found in the N-terminal six-helix bundle facing the cytosol (in), as indicated by an arrow. b The LZNV binding site, marked in (a) by a black box, is displayed enlarged. Side chains undergoing significant conformational changes upon LZNV binding are depicted as sticks and coloured in blue (apo structure) and yellow (LZNV bound structure). The LZNV molecule is displayed in a smaller stick-diameter mode compared to the side chain moieties and coloured in black. Amino acids are labelled in the one-letter code and corresponding TMs are indicated in brackets. View into the PZ pocket location in the apo (c) and LZNV bound structures (d). Side chains from residues forming the PZ pocket are labelled and coloured as in b. Solvent accessible volume is displayed and coloured in grey (c, d).

Fig. 7. Proposed proton-dependent oligopeptide transporter (POT) inhibition mechanism by LZNV.

Cartoon of LZNV (dipeptide amino acid-moiety (AA-moiety): bright pink and Z(NO₂)-moiety: dark pink) approaching a peptide transporter in the outward-open state followed by its partial and full binding. Notice that the PZ pocket is initially absent and forms after binding of the conserved dipeptide backbone to the protein. The Z(NO₂)-moiety bound to the PZ pocket acts like a wedge impeding the transition to the inward-open state. LZNV-POT interactions are indicated by dotted lines. Whereas the grey bar represents the lipid bilayer, the N- and C-terminal bundles are coloured in blue and dark yellow, respectively.