RESUMO
The FapR protein of Bacillus subtilis has been shown to play an important role in membrane lipid homeostasis. FapR acts as a repressor of many genes involved in fatty acid and phospholipid metabolism (the fap regulon). FapR binding to DNA is antagonized by malonyl-CoA, and thus FapR acts as a sensor of the status of fatty acid biosynthesis. However, malonyl-CoA is utilized for fatty acid synthesis only following its conversion to malonyl-ACP, which plays a central role in the initiation and elongation cycles carried out by the type II fatty acid synthase. Using in vitro transcription studies and isothermal titration calorimetry, we show here that malonyl-ACP binds FapR, disrupting the repressor-operator complex with an affinity similar to that of its precursor malonyl-CoA. NMR experiments reveal that there is no protein-protein recognition between ACP and FapR. These findings are consistent with the crystal structure of malonyl-ACP, which shows that the malonyl-phosphopantetheine moiety protrudes away from the protein core and thus can act as an effector ligand. Therefore, FapR regulates the expression of the fap regulon in response to the composition of the malonyl-phosphopantetheine pool. This mechanism ensures that fatty acid biosynthesis in B. subtilis is finely regulated at the transcriptional level by sensing the concentrations of the two first intermediates (malonyl-CoA and malonyl-ACP) in order to balance the production of membrane phospholipids.
Assuntos
Proteína de Transporte de Acila/química , Ácidos Graxos/biossíntese , Proteína de Transporte de Acila/genética , Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Malonil Coenzima A/química , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transcrição GênicaRESUMO
Malonyl-CoA is an essential intermediate in fatty acid synthesis in all living cells. Here we demonstrate a new role for this molecule as a global regulator of lipid homeostasis in Gram-positive bacteria. Using in vitro transcription and binding studies, we demonstrate that malonyl-CoA is a direct and specific inducer of Bacillus subtilis FapR, a conserved transcriptional repressor that regulates the expression of several genes involved in bacterial fatty acid and phospholipid synthesis. The crystal structure of the effector-binding domain of FapR reveals a homodimeric protein with a thioesterase-like 'hot-dog' fold. Binding of malonyl-CoA promotes a disorder-to-order transition, which transforms an open ligand-binding groove into a long tunnel occupied by the effector molecule in the complex. This ligand-induced modification propagates to the helix-turn-helix motifs, impairing their productive association for DNA binding. Structure-based mutations that disrupt the FapR-malonyl-CoA interaction prevent DNA-binding regulation and result in a lethal phenotype in B. subtilis, suggesting this homeostatic signaling pathway as a promising target for novel chemotherapeutic agents against Gram-positive pathogens.