Different Conformations Revealed by NMR Underlie Resistance to Ceftazidime/Avibactam and Susceptibility to Meropenem and Imipenem among D179Y Variants of KPC β-Lactamase

Abstract

β-Lactamase-mediated resistance to ceftazidime-avibactam (CZA) is a serious limitation in the treatment of Gram-negative bacteria harboring Klebsiella pneumoniae carbapenemase (KPC). Herein, the basis of susceptibility to carbapenems and resistance to ceftazidime (CAZ) and CZA of the D179Y variant of KPC-2 and -3 was explored. First, we determined that resistance to CZA in a laboratory strain of Escherichia coli DH10B was not due to increased expression levels of the variant enzymes, as demonstrated by reverse transcription PCR (RT-PCR). Using timed mass spectrometry, the D179Y variant formed prolonged acyl-enzyme complexes with imipenem (IMI) and meropenem (MEM) in KPC-2 and KPC-3, which could be detected up to 24 h, suggesting that IMI and MEM act as covalent β-lactamase inhibitors more than as substrates for D179Y KPC-2 and -3. This prolonged acyl-enzyme complex of IMI and MEM by D179Y variants was not observed with wild-type (WT) KPCs. CAZ was studied and the D179Y variants also formed acyl-enzyme complexes (1 to 2 h). Thermal denaturation and differential scanning fluorimetry showed that the tyrosine substitution at position 179 destabilized the KPC β-lactamases (KPC-2/3 melting temperature [T_m] of 54 to 55°C versus D179Y T_m of 47.5 to 51°C), and the D179Y protein was 3% disordered compared to KPC-2 at 318 K. Heteronuclear ¹H/¹⁵N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy also revealed that the D179Y variant, compared to KPC-2, is partially disordered. Based upon these observations, we discuss the impact of disordering of the Ω loop as a consequence of the D179Y substitution. These conformational changes and disorder in the overall structure as a result of D179Y contribute to this unanticipated phenotype.

Keywords: D179Y; KPC; KPC D179Y; Klebsiella pneumoniae; antibiotic resistance; beta-lactamases; ceftazidime-avibactam resistance.

FIG 1

Expression of KPC-2 and KPC-3 did not differ significantly from that of D179N and D179Y variants. RT-PCR data for gene expression by E. coli DH10-β in (A) LB medium and (B) LB medium supplemented with ceftazidime (32 μg/mL) are shown. Expression of KPC-2 and KPC-3 was set at 1.0 (normalized to 16S rRNA), and relative expression of variant genes is presented as the mean ± standard deviations. NS, nonsignificant, P > 0.05. *, KPC3/D179Y expression (0.77 ± 0.09 [P = 0.01]) was not considered significantly decreased since the difference relative to that of KPC3 was ≤2-fold (1.3-fold).

FIG 2

Thermal denaturation by differential scanning fluorimetry shows that KPC-2 D179Y (A) and KPC-3 D179Y (B) exhibit lower melting temperatures (T_m) than the corresponding WT enzyme. An analysis to extract the Gibbs free energy of unfolding (Δ_uG) confirms that KPC-2 D179Y (C) and KPC-3 D179Y (D) are thermodynamically less stable than the corresponding WT enzymes. The addition of MEM significantly stabilizes KPC-3 D179Y, as evidenced by increases in T_m and Δ_uG ^⨁. (E) The fraction of folded protein, determined by a sigmoidal Boltzmann fit to differential scanning fluorimetry data, is provided for WT (KPC-2 WT) and D179Y KPC-2 (KPC-2 D179Y) in the apo form, or in the presence of MEM or CAZ. (F) The fraction of folded protein, determined by a sigmoidal Boltzmann fit to differential scanning fluorimetry data, is provide for WT (KPC-3 WT) and D179Y KPC-3 (KPC-3 D179Y) in the apo form, or in the presence of MEM or CAZ.

FIG 3

NMR spectra at 308 K of WT KPC-2 (A) and D179Y KPC-2 (B) are markedly different. Poorer chemical shift dispersion and broadening of resonances for D179Y KPC-2 (B) compared to those for WT KPC-2 (A) suggest that D179Y KPC-2 is partially disordered at 308 K. Blue squares with labels indicate resonance assignments from WT KPC-2 for a selected subset of residues near the active site or Ω loop (A). The corresponding positions in the D179Y KPC-2 spectra are also shown as blue squares (B).

FIG 4

Superimposition of KPC-2 versus KPC-3, both with the D179Y substitutions, shows that the 2 enzymes have similar overall structures (A). However, the D179Y substitution (yellow) disorders the Ω-loop in KPC-2 and KPC-3 (B). The most significant change is the disruption of the ionic bond between E166 and K73, present in KPC-2 WT but not the variant, and the movement of K73 away from E166 (≈3.7 Å) and toward S70, to an H-bond distance (1.6 Å). R164 is shifting with ∼3 Å, and the ionic bond with D179 present in WT is replaced by steric interactions of Y179 with D163 in the variant (B). The catalytic water is displaced from E166-N170, and with the K73 moving away from E166, toward S70 and S130, the electrostatics of the D179Y active site are significantly changed.

FIG 5

KPC-2 (A) versus D179Y (B) models during 500 ps molecular dynamic simulation (MDS). The substitution at position D179 is changing the shape of the loop leading to major interactions being disrupted (an ionic bond D179-R164), with new interactions between Y179-R164 being created. During the KPC-2 MDS, the K73 residue maintains the ionic bond with E166 and is moving away from S70 (from 2.6 to 4 Å). The K73 in the variant is making H-bonds with N132, and the catalytic water is better positioned between E166-N170-S70 in the D179Y variant versus KPC-2 as well (B).

FIG 6

Molecular models of KPC-2 (A) versus D179Y (B) as acyl-enzymes, with MEM bound. The acyl MEM is perfectly positioned into the active site of KPC-2, ready for deacylation. The catalytic water (WAT) is placed between E166 and N170, makes H-bonds with E166-S70, and is readily available for proton transfer. A second WAT is in the proximity of, and ready to form H-bonds with, N132 and E166.

FIG 7

MDS of CAZ docked into active site of KPC-2 D179Y variant as acyl-enzyme complex, with snap shots at different times: t = 0 ps (A) and t = 200 ps (B). We follow the conformational changes and catalytic water movement during the 500 ps MDS. Deacylation water, present in the active site at the beginning of MDS simulation, does not make H-bonds with S70 (A). The H-bond distance between WAT and K73 is preserved for most of the MDS, and the WAT molecule makes/breaks bonds with S70. This supports the hypothesis that K73 can act as a general base by activating the WAT in the hydrolysis of CAZ.