title: Structural characterization of a ligand-bound form of Bacillus subtilis FadR involved in the regulation of fatty acid degradation.
abstract: Bacillus subtilis FadR (FadR(Bs)), a member of the TetR family of bacterial transcriptional regulators, represses five fad operons including 15 genes, most of which are involved in β-oxidation of fatty acids. FadR(Bs) binds to the five FadR(Bs) boxes in the promoter regions and the binding is specifically inhibited by long-chain (C14-C20 ) acyl-CoAs, causing derepression of the fad operons. To elucidate the structural mechanism of this regulator, we have determined the crystal structures of FadR(Bs) proteins prepared with and without stearoyl(C18)-CoA. The crystal structure without adding any ligand molecules unexpectedly includes one small molecule, probably dodecyl(C12)-CoA derived from the Escherichia coli host, in its homodimeric structure. Also, we successfully obtained the structure of the ligand-bound form of the FadR(Bs) dimer by co-crystallization, in which two stearoyl-CoA molecules are accommodated, with the binding mode being essentially equivalent to that of dodecyl-CoA. Although the acyl-chain-binding cavity of FadR(Bs) is mainly hydrophobic, a hydrophilic patch encompasses the C1-C10 carbons of the acyl chain. This accounts for the previous report that the DNA binding of FadR(Bs) is specifically inhibited by the long-chain acyl-CoAs but not by the shorter ones. Structural comparison of the ligand-bound and unliganded subunits of FadR(Bs) revealed three regions around residues 21-31, 61-76, and 106-119 that were substantially changed in response to the ligand binding, and particularly with respect to the movements of Leu108 and Arg109. Site-directed mutagenesis of these residues revealed that Arg109, but not Leu108, is a key residue for maintenance of the DNA-binding affinity of FadR(Bs).
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Fujihashi
authors.forename: Taiga
authors.initials: T
authors.lastname: Nakatani
authors.forename: Kazutake
authors.initials: K
authors.lastname: Hirooka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuoka
authors.forename: Yasutaro
authors.initials: Y
authors.lastname: Fujita
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; C057119:FadR protein, Bacteria; D005227:Fatty Acids; D021122:Protein Subunits; D012097:Repressor Proteins
keywords: TetR family; acyl-CoA; crystal structure; transcriptional regulator; β-oxidation of fatty acids