Random and site-specific mutagenesis of theHelicobacter pyloriferric uptake regulator provides insight into Fur structure–function relationships

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Abstract

The ferric uptake regulator (Fur) ofHelicobacter pyloriis a global regulator that is important for colonization and survival within the gastric mucosa.H. pyloriFur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) andapoforms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur andapo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affectedapo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target geneamiE, and theapo-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 (HpFurE5A, HpFurA92T and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3) respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in manganese-substituted andapoconditions byin vitrocross-linking assays, and DNA binding to Fe-Fur andapo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2 and S3 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 howH. pyloriFur regulates gene expression in both the iron-bound andapoforms of the protein.

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