title: Fatty acyl-CoA binding domain of the transcription factor FadR. Characterization by deletion, affinity labeling, and isothermal titration calorimetry.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, New York 12208, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: DiRusso
authors.forename: V
authors.initials: V
authors.lastname: Tsvetnitsky
authors.forename: P
authors.initials: P
authors.lastname: Højrup
authors.forename: J
authors.initials: J
authors.lastname: Knudsen
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D017931:DNA Primers; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D000214:Acyl Coenzyme A; D000345:Affinity Labels; D001426:Bacterial Proteins; D017931:DNA Primers; C057119:FadR protein, Bacteria; D012097:Repressor Proteins; D006639:Histidine