PMID:11581261
| Citation |
Moore, LJ and Kiley, PJ (2001) Characterization of the dimerization domain in the FNR transcription factor. J. Biol. Chem. 276:45744-50 |
|---|---|
| Abstract |
The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) 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 activity in 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 alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-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 O(2) availability. |
| Links |
PubMed Online version:10.1074/jbc.M106569200 |
| Keywords |
Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Chromatography, Gel; Circular Dichroism; Dimerization; Escherichia coli Proteins; 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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