Structural basis for antibiotic transport and inhibition in PepT2, the mammalian proton-coupled peptide transporter

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 mammalian proton-coupled peptide transporter, PepT2, 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 antibiotic recognition and the important role of protonation in drug binding and transport.

Figure 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 DmH⁺ (acidic outside). (B) Chemical structures of cefadroxil, amoxicillin and cloxacillin. (C) IC₅₀ data for the antibiotics and di-alanine peptide. (D) Counterflow assay data showing the ability of only cefadroxil and amoxicillin to drive transport. (E) Solid Support Membrane recordings for the transport of di-alanine peptide, cefadroxil and amoxicillin.

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

(A) Electrostatic surface representation of the cryo-EM structure of PepT2 bound to cefadroxil, highlighting the key structural features of the transporter. Cefadroxil is 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) Equivalent representation for the amoxicillin complex structure.

Figure 3. Analysis of cefadroxil and amoxicillin interactions.

(A) 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. (B)Schematic showing the interactions between PepT2 and cefadroxil. Hydrogen bond donors and acceptors are indicated by arrows, and coloured lines indicate electrostatic interactions. (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. (D) Schematic interaction map between PepT2 and amoxicillin.

Figure 4. 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 three orientations overlaid in the binding site. (B) Zoomed view of the PepT2 binding site, showing the orientation and interactions for pose 1. (C) Pose 2. The cryo-EM density for each pose is shown on the right, contoured at a threshold level ~ 0.22.

Figure 5. 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 1ms-long replicate taken in the last 200ns 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. Protein residue densities were surrounded with black ovals, with the chemical structures of cefadroxil and amoxicillin overlaid for illustration.

Figure 6. Interaction of different beta-lactam antibiotics with PepT2

(A) Counterflow transport assays to discriminate substrates from inhibitors. (B) IC₅₀ values were calculated for transported antibiotics. (C) Classification of the tested beta-lactam antibiotics into substrates or inhibitors.

Figure 7. Structural basis for recognition and transport of beta-lactam antibiotics.

(A) Overlay of the cefadroxil and amoxicillin structures (this study) with the peptide-bound structure of human PepT2 (PDB:7PMX). (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. 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.