Metallo-β-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions

Abstract

A number of multiresistant bacterial pathogens inactivate antibiotics by producing Zn(II)-dependent β-lactamases. We show that metal uptake leading to an active dinuclear enzyme in the periplasmic space of Gram-negative bacteria is ensured by a cysteine residue, an unusual metal ligand in oxidizing environments. Kinetic, structural and affinity data show that such Zn(II)-cysteine interaction is an adaptive trait that tunes the metal binding affinity, thus enabling antibiotic resistance at restrictive Zn(II) concentrations.

Figure 1. The capacity to confer resistance of BcII-Cys221Asp enzyme to E. coli cells is impaired under low Zn^II conditions

The antibiotic sensitivity of E. coli JM109 expressing BcII-Cys221Asp and BcII in the periplasmic space were measured as a function of the Zn^II availability in the growth medium. Colony spots result from serial dilutions of cells transformed with the pKP vector (negative control), pKP-BcII (expressing wild-type BcII), and pKP-BcII- Cys221Asp (expressing the Cys221Asp mutant). Each strain was challenged with ampicillin (32 µg/ml) and cefotaxime (2 µg/ml), under different Zn^II concentrations; namely, low Zn^II (5 µM EDTA), no added Zn^II, and excess Zn^II (250 µM ZnSO₄). These experiments were repeated three times with independent cultures for each condition tested. The length of the white scale bar corresponds to 20 mm.

Figure 2. Structures of the mono-Zn^II and di-Zn^II binding sites of BcII-Cys221Asp enzyme

The metal-binding sites of mono-Zn^II (a), and di-Zn^II (b) BcII-Cys221Asp were solved at 1.71 Å and 1.58 Å resolution, respectively. Electron density maps (2F_O – F_C) are shown for Asp221 and Arg121 sidechains, contoured at 1.5 σ. Numbers indicate Zn^II ions. Dotted lines indicate relevant electrostatic interactions. An acetate ion (ACE) was modeled bound to the di-Zn^II form. Red spheres depict metal-bound water molecules.

Figure 3. The monoCo^II BcII-Cys221Asp is inactive and unable to bind the imipenen substrate

The hydrolysis of imipenem mediated by di-Co^II BcII-Cys221Asp (a), and mono-Co^II BcII-Cys221Asp (b) was detected via photodiode-array stopped-flow kinetic measurements. Spectra represent absorbance for a time span of 100 milliseconds (from pink to black). Insets show the same data as difference spectra (ΔA) taking the spectrum obtained after 100 milliseconds as reference. The initial spectrum in (a) exhibits an absorption band at 390 nm due to formation of a reaction intermediate, which decays in time together with ligand-field bands in the 600–650 nm region. The spectrum of the mono-Co^II form (peak at 540 nm) (b) remained unchanged after 50 seconds. Reaction medium was 100 mM Hepes pH 7.5, 200 mM NaCl, at 7 °C, with 163 µM for both mono- and di-Co^II species and an initial imipenem concentration of 2.5 mM.