Structural basis for antibiotic transport and inhibition in PepT2

Abstract

The uptake and elimination of beta-lactam antibiotics in the human body are facilitated by the proton-coupled peptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2). The mechanism by which SLC15 family transporters recognize and discriminate between different drug classes and dietary peptides remains unclear, hampering efforts to improve antibiotic pharmacokinetics through targeted drug design and delivery. Here, we present cryo-EM structures of the proton-coupled peptide transporter, PepT2 from Rattus norvegicus, in complex with the widely used beta-lactam antibiotics cefadroxil, amoxicillin and cloxacillin. Our structures, combined with pharmacophore mapping, molecular dynamics simulations and biochemical assays, establish the mechanism of beta-lactam antibiotic recognition and the important role of protonation in drug binding and transport.

Fig. 1. Functional characterization of beta-lactam transport by PepT2.

a Overview of PepT1 and PepT2 function in the body. Peptides are transported into the cell via PepT1 and PepT2, driven by the inwardly directed proton gradient ΔμH⁺ (acidic outside). b Chemical structures of cefadroxil, amoxicillin and cloxacillin. c Representative IC₅₀ data for the antibiotics and di-alanine peptide (n = 3 biological experiments performed on different days, mean and errors shown are SD). d Counterflow assay data showing the ability of only cefadroxil and amoxicillin to drive transport. LZNP refers to the inhibitor of PepT1 & 2, Lys [Z(NO₂)]-Proline. 0.5 mM of each ligand was used unless stated otherwise. (n = 5 independent experiments performed on different days, mean shown and errors indicate SD). e Representative solid support membrane recordings for the transport of di-alanine peptide, cefadroxil and amoxicillin and the non-transported compounds, alanine and cloxacillin. Created in BioRender. Newstead, S. (2023) BioRender.com/f58m033.

Fig. 2. Cryo-EM structure of antibiotic-PepT2 complexes.

a Electrostatic surface representation of the cryo-EM structure of PepT2 bound to cefadroxil and amoxicillin, highlighting the key structural features of the transporter. Cefadroxil and amoxicillin are shown in a stick representation. The position of the extracellular domain is indicated but not coloured due to it being absent in the deposited models. b The binding site of PepT2 shows the bound cefadroxil antibiotic (yellow sticks) with nearby and interacting side chains. The cryo-EM density is shown in purple, contoured at a threshold of 0.487. Hydrogen bonds are indicated (cyan dashed lines). Side chains and transmembrane helices are labelled. c The binding site of PepT2 shows the bound amoxicillin (cyan sticks). The cryo-EM density is shown in purple, contoured at a threshold of 0.442. Hydrogen bonds are indicated (yellow dashed lines). d Schematic showing the interactions between PepT2 and cefadroxil. Hydrogen bond donors and acceptors are indicated by arrows, and coloured lines indicate electrostatic interactions. Where electrostatic distances exceed 4 Å the interactions are indicated by positive and negative signs. Distances (Å) are calculated from heavy atoms. e Schematic interaction map between PepT2 and amoxicillin.

Fig. 3. The cloxacillin-bound structure of PepT2 reveals an inhibition mechanism.

a Electrostatic surface representation of the cryo-EM structure of PepT2 bound to cloxacillin, showing the two orientations overlaid in the binding site. b Zoomed view of the PepT2 binding site, showing the orientation and interactions for pose 1. Side chains and transmembrane helices are labelled. c Pose 2. The cryo-EM density for each pose is shown on the right, contoured at a threshold level ~ 0.22. Side chains and transmembrane helices are labelled.

Fig. 4. Protonation of Glu56 promotes ligand recognition via the E⁵³xxER motif.

a Microsecond-long unbiased molecular dynamics (MD) simulations starting at the cefadroxil and amoxicillin cryo-EM models, using 6 replicates for each condition (standard protonation state, E53 protonated and E56 protonated). Histograms of the pooled trajectories of each condition are shown for the drug N-terminus (amino N) – E622 (Cδ) distance (upper block) and drug carboxyl carbon – R57 (Cζ) distance (lower block). b Structural overlay of an example frame from a 1μs-long replicate taken in the last 200 ns with the cryo-EM structure of the cefadroxil complex. c Pooled replicate trajectories (6 μs total, same trajectories as in part a, cefadroxil and amoxicillin standard protonation states and E56 protonated, respectively) projected onto the plane spanned by the Cα atoms of E622, R57 and W313. Positions of E622, R57, the drug N- and C-terminus (all as defined above) and E56 (Cδ) are shown as 2D-histograms in the plane. Colours correspond to the different charges shown (red – negative; blue – positive). Protein residue densities were surrounded with black ovals, with the chemical structures of cefadroxil and amoxicillin overlaid for illustration.

Fig. 5. Interaction of different beta-lactam antibiotics with PepT2.

a Counterflow transport assays to discriminate substrates from inhibitors. (n = 4 independent biological experiments performed on different days and mean shown and errors indicate SD). b Representative IC₅₀ data for the antibiotics cefaclor, moxalactam and ampicillin. c Classification of the tested beta-lactam antibiotics into substrates or inhibitors.

Fig. 6. Pharmacophore model and transport mechanism for beta-lactam antibiotics.

a Overlay of the cefadroxil and amoxicillin structures (this study) with the peptide-bound structure of human PepT1 (PDB:7PMX). Conserved anchor points for the substrate amino and carboxylate groups are shown with conserved pockets within the transporter binding site. b horizontal view of the binding site illustrating the difference in binding pose between cefadroxil and amoxicillin. c Initial steps in beta-lactam transport into the cell. Step 1 shows cefadroxil binding via the primary amine group to Glu622. Step 2 illustrates the movement of protons from His87 (TM2) to Glu56 (TM1), which releases Arg57 to clamp cefadroxil in the binding site. d Final step in drug release into the cell. Step 3 illustrates the protonation of Glu622, which weakens the interaction with the primary amine on the beta-lactam in the inward facing state, here modelled using AlphaFold (AF-Q63424-F1). Step 4 shows the deprotonation of Glu56, which results in Arg57 swinging back to engage the E⁵³xxER motif. Step 5 is the release of the drug into the cytoplasm with two protons.