Characterization of the Dimerization Domain in the FNR Transcription Factor

The global anaerobic regulator FNR fromEscherichia coli is a dimeric Fe-S protein that is inactivated by O2 through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanin...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-12, Vol.276 (49), p.45744-45750
Main Authors: Moore, Laura J., Kiley, Patricia J.
Format: Article
Language:English
Subjects:
alanine scanning mutagenesis
Amino Acid Sequence
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Chromatography, Gel
Circular Dichroism
Dimerization
Escherichia coli
Escherichia coli Proteins
FNR protein
Iron-Sulfur Proteins - chemistry
Iron-Sulfur Proteins - genetics
Iron-Sulfur Proteins - metabolism
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Sequence Homology, Amino Acid
Transcription Factors - chemistry
Transcription Factors - genetics
Transcription Factors - metabolism
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Summary:The global anaerobic regulator FNR fromEscherichia coli is a dimeric Fe-S protein that is inactivated by O2 through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activityin vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between α-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an α-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O2 availability.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M106569200