Cryo-EM characterization of the anydromuropeptide permease AmpG central to bacterial fitness and β-lactam antibiotic resistance

Abstract

Bacteria invest significant resources into the continuous creation and tailoring of their essential protective peptidoglycan (PG) cell wall. Several soluble PG biosynthesis products in the periplasm are transported to the cytosol for recycling, leading to enhanced bacterial fitness. GlcNAc-1,6-anhydroMurNAc and peptide variants are transported by the essential major facilitator superfamily importer AmpG in Gram-negative pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Accumulation of GlcNAc-1,6-anhydroMurNAc-pentapeptides also results from β-lactam antibiotic induced cell wall damage. In some species, these products upregulate the β-lactamase AmpC, which hydrolyzes β-lactams to allow for bacterial survival and drug-resistant infections. Here, we have used cryo-electron microscopy and chemical synthesis of substrates in an integrated structural, biochemical, and cellular analysis of AmpG. We show how AmpG accommodates the large GlcNAc-1,6-anhydroMurNAc peptides, including a unique hydrophobic vestibule to the substrate binding cavity, and characterize residues involved in binding that inform the mechanism of proton-mediated transport.

Fig. 1. Role of AmpG in peptidoglycan recycling and antibiotic resistance.

a Under physiological conditions, mature PG is processed into GlcNAc-1,6-anhydroMurNAc peptides (primarily tetrapeptides) that are transported across the bacterial inner membrane to the cytosol by AmpG for recycling. An intermediate in this normal PG biosynthetic pathway, UDP-MurNAc-pentapeptide, binds to AmpR, which represses production of the class C β-lactamase AmpC. However, in the presence of β-lactams, inhibition of PBPs leads to an accumulation of hydrolyzed PG pentapeptide fragments, which are transported to the cytosol by AmpG, converted to 1,6-anhydroMurNAc-pentapeptides by NagZ, bind AmpR and induce expression of ampC. Secreted AmpC hydrolyzes β-lactams, providing antibiotic resistance. b Chemically synthesized GlcNAc-1,6-anhydroMurNAc derivatives made for this study.

Fig. 2. Overall architecture of wild-type E. coli AmpG.

a AmpG constructs used in this study. b Cryo-EM reconstruction of E. coli AmpG colored by local resolution. c Secondary structure topology of AmpG. d Structure of AmpG in ribbon colored as in c. DDM (light blue) and PE (sea green) shown as sticks with cpk coloring. e Electrostatic surface map (calculated with Coulombic electrostatic potential) of AmpG. f Conserved residue surface map (calculated with Consurf) of AmpG.

Fig. 3. Conserved residues in E. coli AmpG.

a Sequence conservation logo created using 2936 sequences identified from EVcouplings with a bitscore of 0.7. Plotted in WebLogo3 with key motifs and features annotated (see methods). b Binding cavity interactions with the sugar moiety of DDM. c Conserved titratable residues on the C-terminal helical repeat. Glu326 and Asp238 form a conserved carboxyl-carboxylate pair coordinated next to Lys235. d Conserved titratable residues on the N-terminal helical repeat. Motif A Asp70 coordinated by Arg79 and Arg80 interacting with TM11 helix dipole and substrate binding site Lys132, Asp129, and Asp125. e “Foot-in-the-door” motif, which stabilizes the C-terminal helical bundle and helps create the hydrophobic vestibule.

Fig. 4. Substrate binding cavity of outward facing E. coli AmpG.

a AmpG motif A stabilizes the outward conformation with the Asp70 carboxylate forming an electrostatic interaction with the N-terminal helix dipole of TM11, likely enhanced by coordination with Arg79, Arg80, and Asp134. Colored as in Fig. 1d, with mentioned residues marked by *. b Conserved Trp65 and Trp83 in the motif A adaption stabilize the kink in TM2, creating an expanded binding cavity and positioning key residues e.g., Tyr59 and Lys62 for substrate binding. The observed PE lipid extends across the aromatic and nonpolar residues on TM2 and TM4. c Electrostatic surface of the periplasmic substrate binding cavity of AmpG with bound DDM. The maltose sits near an electronegative pocket arising from conserved Asp125 and Asp134, while the hydrophobic acyl chain points into the hydrophobic vestibule between the MFS helical bundle and additional C-terminal helices TM13 and 14. d AmpG substrate binding cavity colored by hydrophobicity. Side chains of disaccharide binding residues shown as sticks and labeled. DDM and lipid are shown as in a. e Density in the binding cavity of AmpG of the modeled bound DDM. f Density consistent with a water coordinated between Lys62, Asp125, and Asp129.

Fig. 5. Modeling of the inward-open state of AmpG.

AmpG in the experimental outward open conformation (a) compared to the AlphaFold3 modeled inward open conformation (b), shown with Coulombic electrostatic potential surface (above) calculated with ChimeraX^, and evolutionary coupled sets of resides (below). Residue pairs on the periplasmic side of the protein, Lys47 and Glu377 (green), are strongly coupled. They are separated ~30 Å in the outward structure but form close contacts in the inward model. At the cytoplasmic side, Asp134 and Ser350 (gold) are also evolutionarily coupled. c Interaction of evolutionary coupled residues E377 and K47 in the modeled inward state. d Structural alignment of N- and C-terminal helical bundles of the experimental outward and modeled inward (gray) states. TM1-TM6 (residues 1–195) RMSD was 1.7 Å, and TM7-TM12 (residues 223-413) RMSD was 1.1 Å.

Fig. 6. Cellular AmpG assays.

a Effect of cefoxitin on growth of a P. aeruginosa PAO1 ampG knockout complemented with E. coli AmpG on low-copy plasmid pHERD30T. Leaky (uninduced) expression of WT ampG (green up-arrow) restored growth to WT levels, while K62A (orange diamond) or Y152A (blue square) mutants had the same expression as empty control (gray down-arrow). Induction of expression with 0.1% arabinose partially restored growth of Y152A but not K62A. b P. aeruginosa AmpG mutants tested as in a. K66A and Y159A affected growth similarly to the ampG knockout (coloring as in a). On induction with 0.1% arabinose, growth of K66A and Y159A was partially restored to varying degrees. c Growth of P. aeruginosa AmpG mutants as in b at varying levels of arabinose induction (gray down-arrow uninduced, blue circle 0.1%, green up-arrow 0.2%, orange diamond 0.5%). Both K66A and Y159A show a concentration-dependent increase in growth on induction, with the response much higher for Y159A. d Identification of a D74N mutant that rescues growth of the P. aeruginosa AmpG K66A mutant. (coloring as in a, blue square represents K66A/D74N). Experiments were performed in technical triplicate and biological duplicate with a representative shown. Data points correspond to technical replicates, with error bands representing SD.

Fig. 7. Proposed AmpG transport mechanism.

a Substrate binding (GlcNAc-1,6-anhydroMurNAc with R representing -OH, tri, tetra, and pentapeptide chains; purple) and protonation of conserved acidic residues are proposed to result in a rigid body conformational shift of AmpG. Motif A Asp70 interacts with the TM11 helix dipole to stabilize the outward open state. The disruption of this interaction e.g., by protonation is proposed to be a key part of the switch to the inward conformation. Positions of conserved titratable residues shown. Dotted arrows suggest a proposed flow of proton transport. Evolutionary coupled residues Lys47 and Glu377 are over 30 Å apart in the outward model but come together to interact in the inward conformation. A potential role of lipid(s) in this mechanism, as suggested by the observed ordered PE, is yet to be determined. b Conserved titratable residues as in a shown as spheres. c Experimentally determined outward-open structure of AmpG. A GlcNAc-1,6-anhydroMurNAc substrate is shown in sticks. d Modeled inward conformation.