Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and larg

The structures of the <i>Sa</i>FapR homodimer in the absence of ligands display distinctive features of either the malonyl-CoA-bound or the DNA-bound forms of the repressor.

<p>(<b>A</b>) Structural superposition of the ligand-free repressor in two different crystal forms (green and cyan) with the malonyl-CoA-bound form (yellow), revealing a similar quaternary organization. Bound malonyl-CoA is shown as solid spheres. Note that helix α_L and its attached DBD are flexible (not modeled) in one monomer of the ligand-free repressor (in cyan, at right). (<b>B</b>) Superposition of this same monomer (magenta) with an equivalent subunit from the malonyl-CoA-bound (cyan) and the DNA-bound (yellow) forms of the repressor. The EBD region (grey molecular surface) is identical for all three monomers. The loop connecting helix α_L with the first β-strand of the EBD in the ligand-free subunit (residues 72–76) has the same conformation as observed in the DNA-bound structure. In both panels, the arrow indicates the first visible residue (Ser72) of the subunit with a disordered helix α_L.</p

Data collection, phasing and refinement statistics.

<p>Values in parentheses are for highest-resolution shell.</p>1<p>According to the MolProbity server <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003108#ppat.1003108-Davis1" target="_blank">[53]</a>.</p

Overall structure of the <i>Sa</i>FapR-operator complex.

<p>(<b>A</b>) Surface representation of the DNA operator (in red) with two bound FapR homodimers looking down the non-crystallographic two-fold symmetry axis. For one homodimeric repressor (in yellow and orange) the DNA-binding domains (DBDs), the linker helix α_L and the dimeric effector-binding domain (DBD) are indicated. (<b>B</b>) ITC study of <i>Sa</i>FapR binding to the P<i>fapR</i> operator at 25°C. The top panel shows the raw heat signal for 6 µl injections of a 68 µM solution of <i>Sa</i>FapR dimer into a 4 µM solution of the 40 bp DNA oligonucleotide (curve 1 obtained by subtraction of the <i>Sa</i>FapR dilution energy curve 3 from the raw titration curve 2). The bottom panel shows the integrated injection heats after normalization fitted with a sequential binding model. Two <i>Sa</i>FapR dimers bind the operator, with parameters (<i>K_d</i>_,I = 0.5±0.1 nM, <i>ΔH</i>°_I = −22.5±0.2 kcal/mol, <i>TΔS</i>°_I = −9.8±0.2, kcal/mol) and (<i>K_d</i>_,II = 51±8 nM, <i>ΔH</i>°_II = −6.95±0.2 kcal/mol, <i>TΔS</i>°_II = 3.0±0.3 kcal/mol).</p

Overall structures of the malonyl-CoA-bound forms of <i>Sa</i>FapR.

<p>(<b>A</b>) Cartoon showing the structure of the <i>Sa</i>FapR-malonyl-CoA homodimer in two different views. The first protomer is shown in green; the second protomer is shown in blue (the helix-turn-helix motif - in dark blue - was partially visible in the electron density map but was not included in the final model due to high protein mobility). Bound malonyl-CoA is shown in surface representation. (<b>B</b>) Closer view of the interactions between the central hot-dog fold (electrostatic surface representation) and the linker helix (green). Hydrophobic side chains involved in inter-domain interactions are labeled. (<b>C</b>) Electron density map of malonyl-CoA and protein-ligand interactions. Hydrogen bonds are indicated by dashed lines and protein residues from each protomer are colored green and yellow respectively.</p

<i>Sa</i>FapR-DNA interactions.

<p>(<b>A</b>) Promoter recognition by the <i>Sa</i>FapR DNA-binding domain. Protein residues making hydrogen-bonding interactions with specific bases (Gln41, Arg56) or with the phosphate backbone are indicated. The DNA double-helix is depicted in solvent accessible surface representation and colored according to the mapped electrostatic potential (negative charge in red, positive in blue). (<b>B</b>) Schematic representation of protein-DNA hydrogen-bonding interactions for one FapR homodimer. Protein residues involved in base-specific hydrogen-bonding interactions are colored red and those involved in phosphate hydrogen-bonding interactions are blue; the specifically recognized bases are orange. In the crystal structure, the DNA duplex exists 50∶50 in the two possible orientations, and the figure shows the 5′ to 3′ sequence covering the 17 bp palindromic sequence (−8 to +8) conserved in promoters of the <i>fap</i> regulon. The non-crystallographic two-fold axis relating the two FapR homodimers in the crystal structure is indicated by the green arrow. (<b>C</b>) Schematic view of the DNA conformation within the FapR–DNA complex. Each base pair is represented by a single block, and the dark-shaded side indicates the minor groove. The actual base-step parameters are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003108#ppat.1003108.s009" target="_blank">Table S2</a>.</p

Expression of the <i>Sa</i>FapR_{G11V, L132W} superrepresor is lethal for <i>S. aureus</i> and fatty acid supplementation cannot overcome growth inhibition.

<p>The figure shows the growth of strain RN4220ΔfapR expressing <i>Sa</i>FapR_{G11V, L132W} (REH117) or <i>Sa</i>FapR_WT (REH118) under the tight inducible P<i>spacOid</i> promoter on THA plates in the absence or presence of 10 mM IPTG. Identical results were obtained for strain REH117 growing in 10 mM IPTG when supplemented with either Tween80 (0.1%) or 500 µM of the following fatty acids: palmitic acid (16∶0), oleic acid (18∶1), 16∶0+18∶1, anteiso 17∶0 (a17∶0), a15∶0+a17∶0, or 18∶0+a17∶0.</p

Structural basis of lipid biosynthesis regulation in Gram-positive bacteria

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