Crystal structure of the class A extended-spectrum β-lactamase CTX-M-96 in complex with relebactam at 1.03 Angstrom resolution

Abstract

The use of β-lactam/β-lactamase inhibitors constitutes an important strategy to counteract β-lactamases in multidrug-resistant (MDR) Gram-negative bacteria. Recent reports have described ceftazidime-/avibactam-resistant isolates producing CTX-M variants with different amino acid substitutions (e.g., P167S, L169Q, and S130G). Relebactam (REL) combined with imipenem has proved very effective against Enterobacterales producing ESBLs, serine-carbapenemases, and AmpCs. Herein, we evaluated the inhibitory efficacy of REL against CTX-M-96, a CTX-M-15-type variant. The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL was covalently bound to the S70-Oγ atom upon cleavage of the C7-N6 bond. Compared with apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.81 Å), making the E166 and N170 side chains shift to create a proper hydrogen bonding network. Binding of REL also disturbs the hydrophobic patch formed by Y105, P107, and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 sidechain is also affected by the piperidine ring. Therefore, differences in the kinetic behavior of REL against class A β-lactamases seem to rely, at least in part, on differences in the residues being involved in the association and stabilization of the inhibitor before hydrolysis. Our data provide the biochemical and structural basis for REL effectiveness against CTX-M-producing Gram-negative pathogens and essential details for further DBO design. Imipenem/REL remains an important choice for dealing with isolates co-producing CTX-M with other β-lactamases.

Keywords: 1,4-diazabicyclooctane; DBO; ESBL; X-ray crystallography; ceftazidimase; enzyme kinetics.

Fig 1

Left: mF₀ –D F_c map electron density map contoured at 1 σ showing relebactam (green mesh) bound to the active site of CTX-M-96. The gray mesh was around the most important residues within the active site, and the deacylating water (DAW) molecule. Right: interaction of relebactam (orange sticks) in the active site of CTX-M-96 (purple sticks), showing the main amino acid residues involved in the stabilizing hydrogen bonds network (black dashed lines), and the deacylation water molecule (DAW; magenta sphere). Other water molecules involved in the network are shown as red spheres.

Fig 2

Computational analysis of the hydrophobic clusters in CTX-M-96 compared with KPC-2 and L2. (a) Amino acid sequence alignment shows the location of the hydrophobic residues involved in the predicted clusters. The color code corresponds to the spatial location of each cluster (see panels b and c). The active site conserved residues are shown in black boxes, and the position of the main secondary structure domains is depicted at the top of the alignment. (b) The spatial coverage of each hydrophobic cluster is shown in different colors. The residue at position 105 is shown as a reference. (c) Area covered by each hydrophobic cluster and number of contacts among the residues in that patch.

Fig 3

Left panel: view of the active site of class A β-lactamases showing the main structural perturbations upon binding of REL. Differential interactions are shown as dashed lines, red for hydrogen bonds, and purple for possible hydrophobic interactions. Color codes: blue/orange (CTX-M-96/REL; PDB 8EHH, this study); green tones (CTX-M-15/REL; PDB 6QW8); yellow tones (KPC-2/REL; PDB 6QW9); and pink/purple (L2/REL; PDB 6QW7). Right panel: comparative interactions of different DBO inhibitors with CTX-M-96. For simplicity, only the amino acid residues creating differential interactions are shown in colors matching the color assigned to the different DBOs: yellow for avibactam (AVI), pink for zidebactam (ZID), and turquoise/cyan for nacubactam (NAC). CTX-M-96 backbone for the complex with REL (orange) is shown in blue, as in the other figures.