Characterization of the dimerization domain in the FNR transcription factor

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] clust er and conversion to a monomeric form. As a first step in elucidating the m olecular interactions that control FNR dimerization, we have performed alan ine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Me t-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 clu ster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A , and FNR-I158A, showed that the Ala substitutions produced specific defect s in dimerization. Because hydrophobic side chains are known to stabilize s ubunit-subunit interactions between a-helices, we propose that Met-147, Ile -151, and Ile-158 lie on the same face of an a-helix that constitutes a dim erization interface. This alignment would also position Arg-140, Met-144, a nd 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 repla cing Asp-154 with Ala repaired the defects caused by Ala substitutions of o ther residues located on the same helical face. These data also suggest tha t Asp-154 has an inhibitory effect on dimerization, which may be a key elem ent in the control of FNR dimerization by O-2 availability.