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

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Bacterial cells utilize transcriptional regulators to control intracellular metal ion concentrations.
• Zinc uptake regulator (Zur) proteins, like that in Acinetobacter baumannii (AbZur), repress gene transcription when zinc is abundant.
• Zur proteins are typically homodimeric with multiple metal-binding sites.

Purpose of the Study:

• To elucidate the metal-binding and allosteric regulation mechanisms of AbZur.
• To identify the specific residues involved in metal coordination and DNA binding.
• To understand the structural basis of AbZur's conformational changes upon metal binding.

Main Methods:

• Cadmium(II) X-ray absorption spectroscopy (XAS) to probe metal coordination.
• Site-directed mutagenesis to investigate the role of specific amino acid residues.
• In vitro transcription assays to measure DNA binding affinity.
• Small-angle X-ray scattering (SAXS) to study conformational changes.

Main Results:

• AbZur possesses a regulatory metal site involving H89, C100, H107, and E122.
• The zinc binding affinity (KZn) for the allosteric site is high (6.0 × 10^12 M^-1).
• Cysteine 100 (C100) is critical for tight DNA binding and allosteric switching, as mutations abolish these functions.
• Wild-type AbZur undergoes conformational changes (allosteric switching) upon metal binding, unlike the C100S mutant.

Conclusions:

• The integrity of C100 is essential for AbZur's allosteric regulation and high-affinity DNA binding.
• Allosteric switching involves a protomer rotation/translation dependent on C100.
• These findings provide insights into the broader mechanisms of Fur family repressors.