Characterization of the Metal Receptor Sites in Escherichia coli Zur, an Ultrasensitive Zinc(II) Metalloregulatory Protein

Abstract

The Escherichia coli Zur protein is a Fur homologue that regulates expression of Zn(II) uptake systems. The zinc-loaded form of Zur is proposed to bind DNA and repress transcription of the znuABC genes. Recent in vitro data indicate that the transcriptional activity of Zur is half-maximal when free Zn(II) concentrations are in the sub-femtomolar range, making it the most sensitive Zn(II) metalloregulatory protein reported to date. Previous results indicate that Zur binds at least one zinc; however, little else is known about Zn(II) binding. We have purified E. coli Zur to homogeneity and found that it has two Zn(II) binding sites per monomer with different coordination environments. Using Zn(II) binding assays, ICP-AES analysis, and Zn EXAFS analysis, we show that one zinc is tightly bound in an S(3)(N/O) coordination environment. Both Co(II) and Zn(II) were substituted into the second metal binding site and probed by EXAFS and UV-visible absorption spectroscopy. These studies indicate that Co(II) is bound in an S(N/O)(3) coordination environment with tetrahedral geometry. The Zn(II) EXAFS of Zn(2)Zur, which is consistent with the results for both sites, indicates an average coordination environment of S(2)(N/O)(2), presumably due to one S(N/O)(3) site and one S(3)(N/O) site. These studies reveal the coordination environments that confer such exceptional zinc sensitivity and may provide the foundation for understanding the molecular basis of metal ion selectivity. A comparison of the metal binding sites in Zur with its Fe(II)-sensing homologue Fur provides clues as to why these two proteins with similar structures respond to two very different metal ions.

Deregulation of transition metals homeostasis is a key feature of cadmium toxicity in Salmonella

“…For example, we hypothesize that cadmium could induce the znuABC operon by replacing zinc in metal-binding sites of Zur, thereby affecting its transcriptional activity. In fact, it has already shown that metal replacement in DNAbinding proteins can alter their substrate specificity (Predki and Sarkar 1994) and that the substitution of zinc with cobalt in Zur is associated to a change in the metal coordination environment (Outten et al 2001).…”

The Fur Homologue inBorrelia burgdorferi

“…However, one cysteine that is conserved among the Zur proteins is absent from the B. burgdorferi Fur. E. coli Zur has two metal binding sites that bind zinc in part through coordination with cysteines (41). Which cysteines are involved is not yet known; however, a cysteine uniquely conserved among the Zur proteins seems a likely candidate.…”

Nutrient-gene interactions: a single nutrient and hundreds of target genes

“…In Escherichia coli (Brocklehurst et al, 1999;Outten et al, 2001), Bacillus subtilis (Gaballa and Helmann, 1998;Gaballa et al, 2002) and Saccharomyces cerevisiae (Zhao and Eide, 1997;Jamison McDaniels et al, 1999;Daniels et al, 2002;Bird et al, 2003) a large number of genes were identified encoding metallo-regulatory proteins that act as zinc sensors and transcriptional activators and/or repressors. Zinc finger-containing transcription factors not only regulate 'zinc-sensitive' genes, but also control their own transcription through a positive autoregulatory mechanism (Lyons et al, 2000).…”