Figures and data
Structural conformations of hPepT2.
(a) The structural topology of hPepT2. (b) The structural model of hPepT2 built with the AlphaFold approach. (c) The structural model of hPepT2 built with the homologous modeling method. The three-dimensional structure of rat PepT2 (PDB code: 7NQK) is used as the structural template. (d) The residues that are used to define the geometry of the channel of hPepT2. (e) The distances between the selected residues and representative structural snapshots are analyzed based on MD simulations.
Spontaneous interactions of polymyxin B with hPepT2.
(a) The interaction pathway of polymyxin B with hPepT2. The spatial locations of polymyxin B molecules are represented with colored spheres. The color spectrum from cyan to red indicates the simulation continuity from 0 to 3 μs. (b) The equilibrated conformation of the hPepT2-polymyxin complex according to the proposed pathway. The polymyxin B molecule is shown in brown and blue spheres with its D-Phe6, and N-terminal fatty acyl group labeled.
Key structural moieties and residues involved in the interaction of polymyxin B with hPepT2.
The surface potential of hPepT2 is shown with red color indicating negative potential and blue color indicating positive potential.
Interaction energy between hPepT2 and each residue of polymyxin B.
The interaction energy was calculated using four MD simulation replicates. Note: Coul: electrostatic interaction; VDW: hydrophobic interaction.
Uptake of 3H-Glycosarcosine and polymyxin B fluorescence probe MIPS-9541 by hPepT2 and its mutants.
HEK293 cells were transfected with hPepT2 and its mutant constructs. (a) Uptake of [3H]-Gly-Sar (5 µM) by hPepT2 or its mutants. (b) Uptake of MIPS-9541 (10 µM) by hPepT2 or its mutants. Data are presented as mean ± SD. All experiments were performed in triplicate independently. ***p < 0.001 vs. control by Welch’s t-test.
Uptake of the polymyxin B fluorescence probe MIPS-9541 in HEK293 cells overexpressing hPepT2 or its mutants assessed by fluorescence imaging.
Data are presented as mean ± SD. All experiments were performed in triplicate independently. Note: WT: wild type; NC: negative control.
Transporter kinetic parameters of MIPS-9541 uptake by hPepT2 and its mutants.
Total cell and cell surface expression of hPepT2-c-Flag and its mutants.
HEK293 cells were transfected with hPepT2-c-Flag and its mutant constructs. (a) Cells were lysed and subjected to SDS-electrophoresis. Immunoblots were probed with anti-Flag antibody followed by probing with an anti-actin antibody as a loading control. Representative images of each hPepT2 mutant are shown. (b) Cell surface proteins were labelled with NHS-ss-Biotin and then pulled down with Streptavidin-agarose beads. Biotin-labelled surface protein samples were subjected to SDS-electrophoresis and the immunoblots were probed with anti-Flag antibody. Equal portions of the supernatant samples were separated by SDS-PAGE, and the immunoblots were incubated with anti-actin antibody. Representative images of each hPeT2 mutant are shown. (c) Densitometry analysis of the relative total cell expression of hPepT2 mutants (ratios of Flag/actin). (d) Densitometry analysis of the relative cell surface expression of hPepT2 mutants (ratios of Flag/actin). Data are presented as % of the hPepT2 wildtype control (mean ± SD). Experiments were repeated on three occasions. *p < 0.05; **p < 0.01 vs. wild-type control by unpaired t-test. Note: WT: wild type; NC: vector-transfected negative control.
Uptake of the polymyxin B fluorescence probe MIPS-9541 and polymyxin analogues by hPepT2.
The data are expressed as the fold change in the uptake via hPepT2 vs. that of the vector-transfected control. Data are presented as mean ± SD. All experiments were performed in triplicate independently. ***p < 0.001 vs. MIPS-9541 control by Welch’s t-test.
Chemical modifications, minimum inhibitory concentrations and nephrotoxicity of polymyxin B (PMB) and its analogues.
The kinetic parameters and protein expression profiles of hPepT2 mutant constructs.
