Structural basis for feed-forward transcriptional regulation of membrane lipid homeostasis in Staphylococcus aureus

Abstract

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 large (>30 Å) inter-domain movements; however, both conformational states can be populated by the ligand-free repressor species, as confirmed by the structure of SaFapR in two distinct crystal forms. Disruption of the ability of SaFapR to monitor malonyl-CoA compromises cell growth, revealing the essentiality of membrane lipid homeostasis for S. aureus survival and uncovering novel opportunities for the development of antibiotics against this major human pathogen.

Figure 1. Overall structure of the SaFapR-operator complex.

(A) 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) ITC study of SaFapR binding to the PfapR operator at 25°C. The top panel shows the raw heat signal for 6 µl injections of a 68 µM solution of SaFapR dimer into a 4 µM solution of the 40 bp DNA oligonucleotide (curve 1 obtained by subtraction of the SaFapR 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 SaFapR dimers bind the operator, with parameters (K_{d ,I} = 0.5±0.1 nM, ΔH°_I = −22.5±0.2 kcal/mol, TΔS°_I = −9.8±0.2, kcal/mol) and (K_{d ,II} = 51±8 nM, ΔH°_II = −6.95±0.2 kcal/mol, TΔS°_II = 3.0±0.3 kcal/mol).

Figure 2. SaFapR-DNA interactions.

(A) Promoter recognition by the SaFapR 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) 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 fap regulon. The non-crystallographic two-fold axis relating the two FapR homodimers in the crystal structure is indicated by the green arrow. (C) 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 Table S2.

Figure 3. Overall structures of the malonyl-CoA-bound forms of SaFapR.

(A) Cartoon showing the structure of the SaFapR-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) 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. (C) 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.

Figure 4. The structures of the SaFapR homodimer in the absence of ligands display distinctive features of either the malonyl-CoA-bound or the DNA-bound forms of the repressor.

(A) 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) 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.

Figure 5. The transitional switch between the relaxed and tense states of the repressor involves a significant rearrangement of the DBDs.

(A) SaFapR in complex with malonyl-CoA (shown in stick representation), tense state. (B) SaFapR in complex with DNA, relaxed state in which the amphipathic helix α_L from each protomer associates with each other. (C) Superposition of the two conformational states of the repressor illustrating the structural transition. Solvent accessible surfaces are shown in transparent to highlight the DNA-induced dissociation of the invariant effector-binding domain from the DBDs. The molecules are shown in light (relaxed) and dark (tense) grey, except for the helices from one DBD (colored).

Figure 6. Expression of the SaFapR_{G11V, L132W} superrepresor is lethal for S. aureus and fatty acid supplementation cannot overcome growth inhibition.

The figure shows the growth of strain RN4220ΔfapR expressing SaFapR_{G11V, L132W} (REH117) or SaFapR_WT (REH118) under the tight inducible PspacOid 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.