Structural characterization of a ligand-bound form of Bacillus subtilis FadR involved in the regulation of fatty acid degradation.

Bacillus subtilis FadR (FadRBs), 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. FadRBs binds to the five FadRBs 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 FadRBs 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 FadRBs 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 FadRBs 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 FadRBs 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 FadRBs 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 FadRBs. Proteins 2014; 82:1301–1310. © 2013 Wiley Periodicals, Inc.