Random and site‐specific mutagenesis of the H elicobacter pylori ferric uptake regulator provides insight into Fur structure–function relationships

The ferric uptake regulator ( Fur ) of H elicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H . pylori Fur is unique in its ability to activate and repress gene expression in both the iron‐bound ( F e‐ Fur ) and apo forms ( apo ‐ Fur ). In the current study we combined random and site‐specific mutagenesis to identify amino acid residues important for both Fe ‐ Fur and apo ‐ Fur function. We identified 25 mutations that affected Fe ‐ Fur repression and 23 mutations that affected apo ‐ Fur repression, as determined by transcriptional analyses of the Fe ‐ Fur target gene amiE , and the apo ‐ Fur target gene, pfr . In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wild‐type ( HpFurWT ) and three mutant Fur proteins ( HpFurE 5 A , HpFurA 92 T and HpFurH 134 Y ), which represent mutations in the N ‐terminal extension, the regulatory metal binding site ( S 2) and the structural metal binding site ( S 3) respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron‐binding by atomic absorption spectrophotometry, oligomerization in manganese‐substituted and apo conditions by in vitro cross‐linking assays, and DNA binding to F e‐ Fur and apo ‐ Fur target sequences by fluorescence anisotropy. The results showed that the N ‐terminal, S 2 and S 3 regions play distinct roles in terms of Fur structure–function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H . pylori Fur regulates gene expression in both the iron‐bound and apo forms of the protein.