Fatty acyl-CoA binding domain of the transcription factor FadR. Characterization by deletion, affinity labeling, and isothermal titration calorimetry

• Published in: The Journal of biological chemistry Vol. 273; no. 50; pp. 33652 - 33659
• Main Authors: DiRusso, C C, Tsvetnitsky, V, Højrup, P, Knudsen, J
• Format: Journal Article
• Language: English
• Published: United States 11.12.1998
• Subjects: Acyl Coenzyme A - metabolism; Affinity Labels; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Calorimetry; DNA Primers; Escherichia coli; Histidine - metabolism; Molecular Sequence Data; Peptide Mapping; Repressor Proteins - chemistry; Repressor Proteins - metabolism; Sequence Deletion
• ISSN: 0021-9258
• DOI: 10.1074/jbc.273.50.33652

The Escherichia coli transcription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRDelta1-167, with a palmitoyl-CoA analogue, 9-p-azidophenoxy[9-3H]nonanoic acid-CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187-195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRDelta1-167 for acyl-CoA. The binding was characterized by a large negative DeltaH0, -16 to -20 kcal mol-1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, Kd of 45 and 63 nM and 68 and 59 nM, respectively. The Kd for palmitoyl-CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding.