acyl-CoA Binding Domain Research Articles

The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR.

FadR is an acyl-CoA-responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The apo-protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl-COA: The recently described structure of apo-FadR shows a DNA binding domain coupled to an acyl-CoA binding domain with a novel fold, but does not explain how binding of the acyl-CoA effector molecule > 30 A away from the DNA binding site affects transcriptional regulation. Here, we describe the structures of the FadR-operator and FadR- myristoyl-CoA binary complexes. The FadR-DNA complex reveals a novel winged helix-turn-helix protein-DNA interaction, involving sequence-specific contacts from the wing to the minor groove. Binding of acyl-CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5 A. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 A in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl-CoA-responsive regulation.

Topology of hepatic mitochondrial carnitine palmitoyltransferase I.

Our earlier work using intact mitochondria and isolated mitochondrial outer membranes confirms the observations of Murthy and Pande that CPT-I is located on the mitochondrial outer membranes and supports the notion that this enzyme has a malonyl-CoA binding domain facing the cytosol and an acyl-CoA binding domain facing the inter membrane space. Our data also suggests that coenzyme A binds at the active site of CPT-I, as does acyl-CoA, 2-bromopalmitoyl-CoA, and (+)-hemipalmitoylcarnitinium, but malonyl-CoA does not bind at that site. Inhibition of CPT-I at the malonyl-CoA binding site by HPG and Ro 25-0187, which have no CoA moiety, contributes to a resolution of this question in that the CoA itself is not essential for the binding of malonyl-CoA to its regulatory site, but the dicarbonyl function which is present in malonyl-CoA, HPG, and Ro 25-0187 is absolutely essential. Our re-evaluation of the topology of hepatic mitochondrial CPT-I confirms the original observations that this enzyme has at least two different binding domains, one domain binding malonyl-CoA, HPG, and Ro-25-187 and the other domain binding acyl-CoA and other inhibitors of CPT-I. Furthermore, the malonyl-CoA binding domain is exposed to the cytosolic face of the membrane. Our data showing that treatment of the intact mitochondria with trypsin causes release of adenylate kinase which indicates that trypsin has damaged the mitochondrial outer membrane, possibly allowing trypsin to enter the intermembrane space and act on CPT from within the outer membrane. Since trypsin's action is limited to arginine and lysine residues, an alternative explanation could be that the portion of the protein domain responsible for malonyl-CoA inhibition may not contain these residues. The latter explanation is plausible, since malonyl-CoA was able to protect against loss of activity and sensitivity to inhibition, but did not protect against loss of adenylate kinase, suggesting that rupture of the outer membrane is not necessarily related to loss of CPT activity. These results suggest that some protein domain that is necessary for CPT activity is exposed on the outer surface of the outer membranes. Therefore, it seems likely that trypsin would have to be able to hydrolyse protein domains of CPT that are inaccessible to Nagarse and papain.

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.

Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain.

Acyl-CoA binding proteins (ACBPs) are small (ca. 10 kDa) highly-conserved cytosolic proteins that bind long-chain acyl-CoAs. A novel cDNA encoding ACBP1, a predicted membrane protein of 24.1 kDa with an acyl-CoA binding protein domain at its carboxy terminus, was cloned from Arabidopsis thaliana. At this domain, ACBP1 showed 47% amino acid identity to Brassica ACBP and 35% to 40% amino acid identity to yeast, Drosophila, bovine and human ACBPs. Recombinant (His)6-ACBP1 fusion protein was expressed in Escherichia coli and was shown to bind 14[C]oleoyl-CoA. A hydrophobic domain, absent in the 10 kDa ACBPs, was located at the amino terminus of ACBP1. Using antipeptide polyclonal antibodies in western blot analysis, ACBP1 was shown to be a membrane-associated glycosylated protein with an apparent molecular mass of 33 kDa. The ACBP1 protein was also shown to accumulate predominantly in siliques and was localized to the seed within the silique. These results suggest that the biological role of ACBP1 is related to lipid metabolism in the seed, presumably in which acyl-CoA esters are involved. Northern blot analysis showed that the 1.4 kb ACBP1 mRNA was expressed in silique, root, stem, leaf and flower. Results from Southern blot analysis of genomic DNA suggest the presence of at least two genes encoding ACBPs in Arabidopsis.