Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii

Abstract

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when Zn^II is replete and binds more weakly to DNA when Zn^II is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. Cd^II X-ray absorption spectroscopy (XAS) of the Cd₂Zn₂ AbZur metalloderivative with Cd^II bound to the allosteric sites reveals a S(N/O)₃ first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. K_Zn for this allosteric site is 6.0 (±2.2) × 10¹² M^–1 with a functional “division of labor” among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to K_Zn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔG_c, is negative, consistent with a higher K_Zn for the AbZur-DNA complex and defining a bioavailable Zn^II set-point of ≈6 × 10^–14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.