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

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 Cd 2 Zn 2 Ab Zur 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) × 10 12 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 Ab Zur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, Δ G c , is negative, consistent with a higher K Zn for the Ab Zur-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 Ab Zur 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.