A ZnS₄ Structural Zinc Site in the Helicobacter pylori Ferric Uptake Regulator

Abstract

The ferric uptake regulator, Fur, is a global bacterial transcriptional regulator using iron as a cofactor to bind to specific DNA sequences. This paper describes the biochemical characterization of the native ferric uptake regulator from Helicobacter pylori (HpFur): oligomeric state, metal content, and characterization of a structural metal-binding site. HpFur contains six cysteines with two CxxC motifs, which makes it closer to Bacillus subtilis PerR (BsPerR) than to Escherichia coli Fur (EcFur). Chemical modifications of cysteine residues using iodoacetamide followed by mass spectrometry after enzymatic digestion strongly suggest that these two CxxC motifs containing cysteines 102-105 and 142-145 are involved in zinc binding in a ZnS(4) metal site. The other two cysteines (78 and 150) are not essential for DNA binding activity and do not perturb metal binding as demonstrated with the characterization of a FurC78SC150S double mutant. Chelating agent such as EDTA disrupts the dimeric structure into monomer which did not contain zinc anymore. Reconstitution of dimer from monomer requires reduction and Zn(2+) binding. Cadmium(II) substitution allows also dimer formation from monomer, and Cd(II)-substituted FurC78SC150S mutant presents a characteristic absorption of a Cd(II)Cys(4) metal-binding site. These results establish that coordination of the zinc ion in HpFur is ZnCys(4), therefore closer to the zinc site in BsPerR than in EcFur. Furthermore, the redox state of the cysteines and the zinc binding are essential to hold the H. pylori Fur in a dimeric state.

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur

“…The amount of S1-protected band was presented relative to the level in wild-type (M145) cells containing parental vector (pSET162), as an average value from three independent experiments. A B structure of BsPerR and Fur from Helicobacter pylori (HpFur) (27,28). On the other hand, mutations at M-and D-sites did not affect dimerization as well as the characteristics of secondary structure.…”

Novel Insights into Fur Regulation in Helicobacter pylori

“…S1 is a structural metal binding site located near the oligomerization interface that is coordinated by four cysteine residues. In the other Fur structures, disulfide linkages between analogous cysteine residues function in a similar capacity as this metal binding site, making S1 fairly unique to H. pylori (49,165). H.…”

“…S1 is a structural zinc-binding site (10,165) that is coordinated by four cysteine residues (C102, C105, C142, and C145)…”

“…Metallation at this site stabilizes the three β sheets in this region (β3-β5) and is necessary for dimerization (165). S2 is the regulatory binding site; metallation at S2 is responsible for a conformational change in the DNA binding domain that "activates" the protein and facilitates interaction with Fe-Fur target DNA.…”

“…This idea is supported by CD spectroscopy ( Figure 13A), which detected several changes in overall secondary structure, and the oligomerization assays ( Figure 15). As metallation at S1 is required for dimer formation (49,165), the fact that the A92T protein was able to oligomerize in both the iron-substituted and apo forms strongly suggests that the metal ion(s) detected by atomic absorption spectrophotometry occupy S1. On a cellular level, the dramatic reduction in iron binding also explains the constitutive de-repression of amiE as well as the hyper-repression of pfr that was observed by qRT-PCR (Figures 10 and 11).…”