Protein-DNA interactions in the regulation of lipid biosynthesis in Gram positive bacteria

GEORGINA REH,; SCHUJMAN, GE; DE MENDOZA, D

Rosario

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

Transcriptional regulation of bacterial lipid biosynthetic genes is poorly understood at the molecular level. Indeed, the only well documented example is probably that of fabA and fabB, two essential genes for the synthesis of unsaturated fatty acids in Escherichia coli [1]. A major advance in our understanding of the transcriptional control of bacterial lipid synthesis was achieved through the identification of FapR, a global transcriptional repressor that controls the expression of many genes involved in the biosynthesis of lipids (the fap regulon) in Bacillus subtilis (Bs) [2]. FapR has highly conserved homologues in many Gram-positive bacteria, including several human pathogens. As well, in all these organisms the consensus binding sequence of FapR is largely invariant in the putative promoter regions of the fapR gene, indicating that the regulation mechanism observed in Bs is conserved in many other organisms. We have demonstrated that FapR belongs to a new class of bacterial repressors and that malonyl-CoA operates as the direct and specific inducer of FapR-regulated promoters. Sequence analysis suggests that FapR is a two-domain protein with an N-terminal DNA-binding domain connected through a helical linker to a larger C-terminal domain. This second domain has weak sequence identity with thioesterases. This thioesterase-like domain (TLD) of FapR  posseses the effector-binding function, displaying  a ‘hot-dog’ fold, similar to that of several thioesterases known to process acyl-CoA substrates [3]. However, this fold differs from other known bacterial transcriptional regulators. Binding of malonyl-CoA promotes a disorder-to-order transition in the protein that causes the FapR-DNA complex to dissociate or prevents its formation. In the absence of inducer, the FapR model predicts a direct interaction between the two amphipatic linker helices in order to achieve a functional association of the associated HTH motifs for DNA binding. Although we lack the crystal structure of the FapR-DNA complex, to prove the helix-helix interaction we produced three point mutants. One of them, Ile54-Cys, was designed to cross link the two helices through a disulfide bridge, without affecting the protein functionality. The other two, Ile54-Asp and Val57-Pro, were intended to interfere with helix dimerization and DNA-binding capacity. As expected, FapRI54C was found to form a covalent dimer without losing its DNA-binding capability, whereas the introduction of an aspartate at position 54 (FapRI54D) or a proline at position 57 (FapRV57P) precluded a functional helix-helix association and rendered the protein unable to bind DNA. These results strongly support a close spatial proximity of the two linker helices in inducer-free FapR, as predicted by the model. [1] Schujman GE, de Mendoza D., Curr Opin. Microbiol, 2005, 8, 149-153. [2] Schujman GE, Paolleti L, Grossman AD, de Mendoza D., Dev Cell , 2003, 4, 663-672. [3] Dillon SC and Bateman A, BMC Bioinformatics, 2004, 5, 109-122. Acknowledgments: Part of the experiments presented were performed in the laboratories of Drs. P. Alzari and A.Vila