title: Mutagenesis of conserved amino acids of Helicobacter pylori fur reveals residues important for function.
abstract: The ferric uptake regulator (Fur) of the medically important pathogen Helicobacter pylori is unique in that it has been shown to function as a repressor both in the presence of an Fe2+ cofactor and in its apo (non-Fe2+-bound) form. However, virtually nothing is known concerning the amino acid residues that are important for Fur functioning. Therefore, mutations in six conserved amino acid residues of H. pylori Fur were constructed and analyzed for their impact on both iron-bound and apo repression. In addition, accumulation of the mutant proteins, protein secondary structure, DNA binding ability, iron binding capacity, and the ability to form higher-order structures were also examined for each mutant protein. While none of the mutated residues completely abrogated the function of Fur, we were able to identify residues that were critical for both iron-bound and apo-Fur repression. One mutation, V64A, did not alter regulation of any target genes. However, each of the five remaining mutations showed an effect on either iron-bound or apo regulation. Of these, H96A, E110A, and E117A mutations altered iron-bound Fur regulation and were all shown to influence iron binding to different extents. Additionally, the H96A mutation was shown to alter Fur oligomerization, and the E110A mutation was shown to impact oligomerization and DNA binding. Conversely, the H134A mutant exhibited changes in apo-Fur regulation that were the result of alterations in DNA binding. Although the E90A mutant exhibited alterations in apo-Fur regulation, this mutation did not affect any of the assessed protein functions. This study is the first for H. pylori to analyze the roles of specific amino acid residues of Fur in function and continues to highlight the complexity of Fur regulation in this organism.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Heath Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA.
authors.forename: Beth M
authors.initials: BM
authors.lastname: Carpenter
authors.forename: Hanan
authors.initials: H
authors.lastname: Gancz
authors.forename: Stéphane L
authors.initials: SL
authors.lastname: Benoit
authors.forename: Sarah
authors.initials: S
authors.lastname: Evans
authors.forename: Cara H
authors.initials: CH
authors.lastname: Olsen
authors.forename: Sarah L J
authors.initials: SL
authors.lastname: Michel
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Maier
authors.forename: D Scott
authors.initials: DS
authors.lastname: Merrell
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron