Fatty acyl-CoA binding domain of the transcription factor FadR. Characterization by deletion, affinity labeling, and isothermal titration calorimetry.
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.
DiRusso
C C
CC
Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, New York 12208, USA.
Tsvetnitsky
V
V
Højrup
P
P
Knudsen
J
J
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
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D001426:Bacterial Proteins; D017931:DNA Primers; C057119:FadR protein, Bacteria; D012097:Repressor Proteins; D006639:Histidine