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 ZnII is
replete and binds more weakly to DNA when ZnII is limiting.
Previous studies established that Zur proteins are homodimeric and
harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII 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. KZn 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 KZn 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, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable
ZnII 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.