부산대학교 도서관

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 baumanniiZur represses transcription of its regulon when ZnIIis replete and binds more weakly to DNA when ZnIIis limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdIIX-ray absorption spectroscopy (XAS) of the Cd2Zn2AbZur metalloderivative with CdIIbound to the allosteric sites reveals a S(N/O)3first 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. KZnfor this allosteric site is 6.0 (±2.2) × 1012M–1with a functional “division of labor” among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZnthan H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitrotranscription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZnfor the AbZur-DNA complex and defining a bioavailable ZnIIset-point of ≈6 × 10–14M. 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.