Structural basis of lipid biosynthesis regulation in Gram-positive bacteria
2006; Springer Nature; Volume: 25; Issue: 17 Linguagem: Inglês
10.1038/sj.emboj.7601284
ISSN1460-2075
AutoresGustavo E. Schujman, Marcelo E. Guerin, Alejandro Buschiazzo, Francis Schaeffer, Leticia I. Llarrull, Georgina Reh, Alejandro J. Vila, Pedro M. Alzari, Diego de Mendoza,
Tópico(s)Bacteriophages and microbial interactions
ResumoArticle24 August 2006free access Structural basis of lipid biosynthesis regulation in Gram-positive bacteria Gustavo E Schujman Gustavo E Schujman Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Marcelo Guerin Marcelo Guerin Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Alejandro Buschiazzo Alejandro Buschiazzo Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Francis Schaeffer Francis Schaeffer Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Leticia I Llarrull Leticia I Llarrull Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Química Biológica, Área Biofísica, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Georgina Reh Georgina Reh Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Alejandro J Vila Alejandro J Vila Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Química Biológica, Área Biofísica, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Pedro M Alzari Corresponding Author Pedro M Alzari Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Diego de Mendoza Corresponding Author Diego de Mendoza Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Gustavo E Schujman Gustavo E Schujman Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Marcelo Guerin Marcelo Guerin Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Alejandro Buschiazzo Alejandro Buschiazzo Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Francis Schaeffer Francis Schaeffer Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Leticia I Llarrull Leticia I Llarrull Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Química Biológica, Área Biofísica, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Georgina Reh Georgina Reh Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Alejandro J Vila Alejandro J Vila Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Química Biológica, Área Biofísica, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Pedro M Alzari Corresponding Author Pedro M Alzari Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France Search for more papers by this author Diego de Mendoza Corresponding Author Diego de Mendoza Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Search for more papers by this author Author Information Gustavo E Schujman1,2,‡, Marcelo Guerin3,‡, Alejandro Buschiazzo3, Francis Schaeffer3, Leticia I Llarrull1,4, Georgina Reh1,2, Alejandro J Vila1,4, Pedro M Alzari 3 and Diego de Mendoza 1,2 1Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, Rosario, Argentina 2Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina 3Unité de Biochimie Structurale & URA 2185 CNRS, Institut Pasteur, Paris, France 4Departamento de Química Biológica, Área Biofísica, Universidad Nacional de Rosario, Rosario, Argentina ‡These authors contributed equally to this work *Corresponding authors: Unité de Biochimie Structurale, Institut Pasteur, URA 2185 CNRS, 25 rue du Docteur Roux, Paris 75724, France. Tel.: +33 1 4568 8607; Fax: +33 1 4568 8604; E-mail: [email protected], Suipacha 531, Rosario 2000, Argentina. Tel.: +54 341 435 1235 ext 111; Fax: +54 341 439-0465; E-mail: [email protected] The EMBO Journal (2006)25:4074-4083https://doi.org/10.1038/sj.emboj.7601284 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Our understanding of the relationship between metabolic signals and global changes in gene expression is limited by the difficulty in identifying intracellular signaling molecules that interact with key regulatory proteins. This gap is particularly apparent in the control of membrane lipid homeostasis. Fatty acids and their derivatives play essential roles in all living organisms as components of membranes and source of metabolic energy. Biosynthesis of these compounds involves repeated cycles of condensation, reduction and dehydration of carbon–carbon linkages, which are carried out by a single multifunctional polypeptide (type I systems) in higher eukaryotes (Smith et al, 2003) and by discrete proteins (type II systems) in bacterial cells, plant chloroplasts and malaria parasites (White et al, 2005). Despite the complexity of these biosynthetic pathways, biological membranes maintain stable compositions that are characteristic for different organisms, tissues and intracellular organelles. However, the precise homeostatic mechanisms maintaining the concentration of lipids at particular levels are largely unknown. 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 (Lu et al, 2004; Schujman and de Mendoza, 2005). Two transcriptional regulators belonging to the TetR superfamily, the activator FadR and the repressor FabR, were shown to regulate the expression of the fabA and fabB genes. FadR was discovered as a repressor of the β-oxidation regulon (Overath et al, 1969; Dirusso and Nunn, 1985), and subsequently found to also activate fabA and fabB transcription (Henry and Cronan Jr, 1991, 1992; Lu et al, 2004; Schujman and de Mendoza, 2005). Binding of FadR to its DNA operator is antagonized by different long-chain acyl-CoAs (Dirusso et al, 1992; Raman and Dirusso, 1995; Cronan Jr and Subrahmanyam, 1998; Dirusso et al, 1998), in agreement with the structural model (van Aalten et al, 2000, 2001; Xu et al, 2001). The second E. coli regulator, FabR, acts as a repressor in the regulation of fabA and fabB (Zhang et al, 2002), although it is still unclear which signal(s) modulates its DNA binding activity. 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 fatty acids and phospholipids (the fap regulon) in Bacillus subtilis (Schujman et al, 2003). FapR has highly conserved homologs in many Gram-positive bacteria, including several human pathogens (Schujman et al, 2003). 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 B. subtilis is conserved in many other organisms. We demonstrate here that FapR belongs to a new class of bacterial repressors and that malonyl-CoA, an essential intermediate in fatty acid synthesis, operates as the direct and specific inducer of FapR-regulated promoters. The effector-binding domain of FapR was found to display a ‘hot-dog’ fold, similar to that of several thioesterases known to process acyl-CoA substrates but different 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. Finally, we show that site-directed mutations disrupting the FapR–malonyl-CoA interaction result in a lethal phenotype in B. subtilis, validating the structural model and suggesting that this homeostatic signaling pathway could be a target for novel chemotherapeutic agents against Gram-positive pathogens. Results and discussion Malonyl-CoA is a direct and specific regulator of FapR activity Two observations suggested that the intracellular pool of malonyl-CoA might be associated with the regulation of FapR activity. First, expression of the fap regulon is derepressed by antibiotics that inhibit fatty acid biosynthesis (with the concomitant increase in the intracellular levels of malonyl-CoA; Schujman et al, 2001). Second, this upregulation is abolished by precluding the transcription of genes encoding the subunits of the acetyl-CoA carboxylase (ACC), which catalyzes the synthesis of malonyl-CoA (Schujman et al, 2003). A major question is whether malonyl-CoA directly regulates FapR activity or acts as a building block, converted into another product that is the signaling molecule. For the malonate group to be used in fatty acid synthesis, it must be transferred from malonyl-CoA to acyl-carrier-protein by malonyl-CoA:ACP transacylase, the product of the fabD gene (de Mendoza et al, 2002). After conditional inhibition of the B. subtilis fabD gene (Morbidoni et al, 1996), we still observed transcriptional induction of the fap regulon by antibiotics that inhibit the biosynthetic pathway (data not shown), suggesting that malonyl-CoA could be the direct effector of FapR. To prove this hypothesis, we performed in vitro transcription experiments, initiating at fapR (see Materials and methods). A transcript of the expected size (93 nt) was obtained in the absence of FapR, whereas fapR transcription was inhibited in reactions containing the repressor (Figure 1), confirming that FapR represses transcription. To test whether malonyl-CoA relieves FapR-mediated repression, we repeated the experiments in the presence of FapR and malonyl-CoA at concentrations ranging from 15 to 500 μM. As shown in Figure 1, increasing concentrations of malonyl-CoA gradually induced in vitro fapR transcription. Furthermore, this effect is highly specific since several acyl-CoA derivatives related to malonyl-CoA (such as acetyl-CoA, propionyl-CoA, succinyl-CoA and butyryl-CoA), were not able to relieve the inhibitory action of the repressor on fapR transcription (Figure 1). Similar results were obtained by in vitro transcription analysis using the promoters of other genes belonging to the fap regulon, such as fabI and yhdO, which were repressed by FapR and specifically induced by malonyl-CoA (data not shown). Thus, all these experiments prove that FapR is unable to repress transcription of the fap regulon in the presence of malonyl-CoA, and that this molecule operates as a direct and specific inducer of the fap promoters. Figure 1.Effect of malonyl-CoA and short-chain acyl-CoA thioesters on FapR-mediated repression. In vitro transcription was performed with a PfapR promoter fragment (6.4 nM) as the template in the presence of FapR (120 nM) and different concentrations of malonyl-CoA (upper panel) or malonyl-CoA analogs (lower panel). Download figure Download PowerPoint Malonyl-CoA binds FapR and modulates the repressor-operator interaction Sequence analysis suggests that FapR, like many bacterial repressors, is a two-domain protein with an N-terminal DNA-binding domain (DBD) connected through a helical linker to a larger C-terminal domain (Figure 2). This second domain has weak sequence identity with thioesterases, a family of enzymes known to bind and process acyl-CoA substrates (Dillon and Bateman, 2004), suggesting that the thioesterase-like domain (TLD) of FapR might bear the effector-binding function. To prove this hypothesis, we produced two deletion mutants of FapR (FapRΔ43 and FapRΔ67) that contain the TLD domain but lack the DBD (Figure 2). One of them (FapRΔ43) still harbors the linker helix αL. Isothermal titration calorimetric (ITC) measurements demonstrate that malonyl-CoA binds full-length FapR with an affinity constant in the micromolar range (Figure 3A), which is within a physiologically relevant range (Heath et al, 2002). The malonyl-CoA binding site is entirely defined by the C-terminal domain of the protein, since malonyl-CoA also binds FapRΔ43 with a similar affinity constant. In each case, two molecules of inducer bind the protein dimer in independent non-cooperative events, as indicated by a Scatchard plot derived from the thermodynamic data (Supplementary Figure S1). However, the binding of malonyl-CoA to FapR and FapRΔ43 display some significant differences. While effector binding to FapR was largely entropy driven (ΔH°/ΔG°=24%), binding to FapRΔ43 was enthalpy driven (ΔH°/ΔG°=67%) and displayed a three-fold decrease of the heat capacity change (ΔCp) on binding (see Figure 3A, legend). Both the different nature of the binding driving force and the change of ΔCp might be attributed to a different environment of the malonyl-CoA binding-site in the absence of the HTH motif. On the other hand, under the same experimental conditions the shorter construct FapRΔ67 presented a flat binding isotherm at three different temperatures (Figure 3A). A null enthalpy value over a 20° temperature interval strongly argues for a severe decrease of the malonyl-CoA binding affinity upon deletion of the linker helix αL, indicating that this helix must play an important role in the formation of a competent effector-binding site in FapR. Figure 2.Domain organization of B. subtilis FapR and the two deletion mutants used in this work. Download figure Download PowerPoint Figure 3.Malonyl-CoA binds to the C-terminal domain of FapR and modulates DNA-binding activity. (A) Isothermal calorimetric titrations of FapR with malonyl-CoA. The top panel shows the raw heat signal for 10 μl injections of 240 μM malonyl-CoA into solutions of 16 μM FapR, 20 μM FapRΔ43, and 20 μM FapRΔ67, respectively, at 298 K (curves have been offset by 0.3 μcal/s for clarity). The bottom panel shows the transition curves of the three proteins (FapR, Kd=2.4±0.2 μM; FapRΔ43, Kd=7.1±0.9 μM). The inset shows the enthalpy changes for the above reactions as a function of temperature (FapR, ΔH°=−1.8±0.2 kcal mol−1, ΔCp=−103.0±0.3 cal mol−1 K−1; FapRΔ43, ΔH°=−4.8±0.3 kcal mol−1, ΔCp=−31.9±1.1 cal mol−1 K−1). FapRΔ67 displays a flat binding isotherm at all tested temperatures between 288 and 308 K. (B) PfapR operator region. Capital letters indicate bases protected by FapR from DNAseI digestion; lines delimitate protected regions. Bold letters show hypersensitive spots. Italic T indicates the transcription start base. The 17 bp inverted repeat conserved in all operators of the fap regulon (Schujman et al, 2003) is shown in gray and white letters indicate a 5 bp inverted repeat separated by 23 nt. (C) Fluorescence anisotropy changes on addition of FapRWT to 9.5 nM 34 bp F-dsDNA (black circles); on addition of FapRWT to a mixture of 9.5 nM 34 bp F-dsDNA and 307.7 μM malonyl-CoA (black triangles); and on addition of malonyl-CoA (white triangles), acetyl-CoA (white circles) or malonic acid (gray squares) to the previously formed F-dsDNA/FapRWT complex. Download figure Download PowerPoint The DBD of FapR is predicted to contain a typical HTH domain, which displays a conserved sequence motif in the putative DNA-recognition helix. This motif is similar to that of the deoxyribose repressor DeoR family (Aravind et al, 2005), although the FapR family of repressors lacks the β-hairpin characteristic of the DeoR winged domains (Ni et al, 1999). DNAse I footprinting analyses on both strands of the fapR promoter (Supplementary Figure S2) revealed that the repressor protects a 34 bp DNA region composed by the dyad symmetric consensus element flanked by 5′-AATTA-3′ inverted repeats (Figure 3B). ITC measurements at 25°C show that wild-type FapR binds the 34 bp dsDNA with a dissociation constant of 0.2 μM in an enthalpy-driven event (data not shown). To investigate the effect of malonyl-CoA on FapR–DNA interactions, we performed titration experiments adding FapR to fluorescein-labeled 34 bp dsDNA. Direct binding of DNA to FapR is evidenced by an increase of the fluorescence anisotropy (Figure 3C). Addition of malonyl-CoA to the FapR-DNA adduct results in a significant decrease of the fluorescence anisotropy, whereas acetyl-CoA or malonic acid are unable to induce the same effect. Furthermore, attempts to bind DNA in the presence of saturating concentrations of malonyl-CoA result in a modest increase in the fluorescence anisotropy, indicating that DNA binding barely takes place under these conditions (Figure 3C). These experiments confirm that malonyl-CoA is a specific effector molecule that releases FapR from (or prevents binding to) its DNA operator. Structural basis of malonyl-CoA-binding specificity The crystal structure of the C-terminal domain of FapR (FapRΔ67) was determined at 2.5 Å resolution using a combination of MAD and molecular replacement techniques (Table I). The protein is a homodimer that displays the typical ‘hot-dog’ fold characteristic of the thioesterase enzyme family (Leesong et al, 1996; Li et al, 2000). Each monomer folds into an α/β globular domain with a central α-helix (αC) surrounded by a six-stranded antiparallel β-sheet ‘bun’. Topologically, αC is inserted between strands β2 and β3, with the last four β-strands (β3–β6) arranged in a Greek key motif. The dimer is formed by extensive interaction between the β3 strands of both monomeric partners, giving rise to a single inter-monomeric antiparallel β-sheet, which wraps along its concave side both αC helices (Figure 4). In each monomer, the N-terminal residues of FapRΔ67 (up to position 73) are present but disordered in the crystal structure. Figure 4.Overall structure of the FapRΔ43–malonyl-CoA complex. Ribbon model with secondary structure elements (β-strands 1–6, the linker helix αL and central helix αC) indicated for one monomer. Download figure Download PowerPoint Table 1. Data collection, phasing and refinement statistics Data set SeMet-labeled FapR FapRΔ67 FapRΔ43–malonyl-CoA Data collection Resolution (Å)a 40–3.5 (3.69–3.5) 60–2.5 (2.64–2.5) 63.2–3.1 (3.27–3.1) Wavelength (Å) 0.9791 0.9793 0.9755 1.072 0.9794 Measured reflections 22 839 22 837 23 045 74 467 87 334 Multiplicitya 5.8 (5.8) 5.8 (5.7) 5.8 (5.8) 5.0 (4.3) 6.9 (7.2) Completeness (%)a 99.4 (99.4) 99.5 (99.7) 99.3 (99.2) 80.5 (50.0) 100 (100) Rsym (%)a,b, a,b 9.3 (27.5) 9.7 (32.6) 11.5 (40.3) 8.3 (28.7) 7.9 (31.0) 〈I/σ〉a 13.5 (4.9) 12.8 (4.2) 11.3 (3.3) 13.7 (4.8) 19.3 (6.0) Refinement Resolution (Å) 30–2.5 63.2–3.1 Rcryst c (No. refs) 0.221 (14035) 0.187 (11604) Rfree c (No. refs) 0.267 (790) 0.227 (941) R.m.s. bonds (Å) 0.019 0.02 R.m.s. angles (deg) 1.72 2.14 Protein atoms 2964 2238 Water molecules 8 10 Ligand atoms — 64 a Values in parentheses apply to the high resolution shell. b c Rcryst and Rfree were calculated from the working and test reflection sets, respectively. Formation of the FapR dimer buries 1150 Å2 from each monomer surface, which represents 13.5% of the total accessible surface. The protein presents this same oligomeric arrangement in solution, as indicated by studies in solution of full-length FapR and the two deletion mutants (FapRΔ43 and FapRΔ67) using gel filtration (Supplementary Figure S3), dynamic light scattering and glutaraldehyde cross-linking (data not shown) experiments. To further investigate the FapR–malonyl-CoA interactions, we crystallized FapRΔ43 in complex with the effector molecule and determined the structure of the complex at 3.1 Å resolution (Table I). Crystals of this complex belong to a different space group than those of the unliganded proteins FapRΔ43 or FapRΔ67 (see Materials and methods), further suggesting that malonyl-CoA binding induced a conformational change in the protein. Two malonyl-CoA molecules are deeply buried into the globular core of the dimer (Figure 4). A large fraction of the ligand molecules is positioned within equivalent tunnels that run perpendicular to both the extended β-sheet and the central αC helices on either side of the dimer. Instead, the 3′P-nucleoside moiety of the ligand (disordered in the crystal structure) sticks out from the globular core and is exposed to the bulk solvent, as observed in other acyl-CoA-thioesterase complexes (Thoden et al, 2003). The linker helices αL, which were missing in FapRΔ67, are now visible in the structure of the FapRΔ43–malonyl-CoA complex (Figure 4), clearly detached from the TLD core. The malonyl moiety of the ligand is critical for specificity, since analogous acyl-CoA derivatives are significantly less active in transcription experiments (Figure 1) and do not interfere with FapR–DNA binding (Figure 3C). Both the size of the malonyl moiety (to fit within the binding cavity) and the presence of a carboxylate group at position 1 are primary factors influencing effector-binding specificity. The malonyl carboxylate sits on top of the N-terminal end of helix αC, within a hydrophobic pocket defined by residues Leu69′, Val119′, Asn115′, Ser116′, Phe99 and His108 (primed/unprimed numbers indicate residues from each monomer in the homodimer). Upon malonyl-CoA-binding, the guanidinium group of Arg106 moves from its outer position in free FapR towards the hydrophobic pocket, where it stacks against the aromatic ring of Phe99 and forms a salt bridge with the malonyl CO2− group. Interestingly, Phe99 and Asn115′ (H-bonded to the thioester carbonyl oxygen of the malonyl moiety) are largely conserved in the FapR family of bacterial repressors and occupy the equivalent positions of the two catalytic residues in homologous thioesterases (Thoden et al, 2002). Malonyl-CoA binding to FapR induces significant conformational changes The structural comparison of malonyl-CoA-bound and free FapR reveals that an open groove in the ligand-free protein becomes a tunnel enclosing the effector molecule in the complex (Figure 5A). These changes are due to the structuring of three protein loops, which are partially or totally disordered in ligand-free FapR but display a well-defined conformation in the complex (Figure 5B), with no obvious crystal packing interactions that could account for these differences. The open cleft in free FapR facilitates ligand binding, avoiding the costly path of introducing the charged malonate moiety through a pre-existent hydrophobic tunnel (Benning et al, 1998). Figure 5.(A) Molecular surface of the binding site in the ligand-free (left) and ligand-bound (right) FapR structures. Malonyl-CoA (in red) is shown for reference in the ligand-free structure. (B) Formation of the tunnel involves the structuring of three protein loops (A–C) and the C-terminal end of the protein, which are highly mobile or disordered in ligand-free FapR. Download figure Download PowerPoint The critical interaction between the malonyl carboxylate and Arg106 side-chain at the end of loop B triggers the formation of several H-bonding interactions that ultimately stabilize the conformation of loops A–C in the complex (Figure 6). Upon malonyl-CoA binding, the Arg106 guanidinium group gets engaged in electrostatic interactions with both the malonyl CO2− and the main-chain carbonyl of Glu73′ in loop A. The movement of Arg106 allows the rearrangement of the Glu73′ side-chain, which now forms a strong H-bond with Ser100 on loop B. In turn, the new conformation of loop B facilitates the interaction of the Arg101 guanidinium with the main-chain carbonyls of Lys67′ (loop A) and Glu125′ (loop C), further stabilized by the formation of a strong salt bridge between Lys67′ (loop A) and Asp123′ (loop C). This closure event extends like a ‘zipper’ towards the N-terminus of loop A, involving the formation of additional H-bonds, up to the linker helix αL connecting the TLD and DBD domains. Additional interactions of the ligand phosphates with Tyr183 and Lys186 contribute to stabilize the C-terminal segment of the protein, which is also disordered in ligand-free FapR but makes contacts with loop C residues in the complex. Figure 6.Stereoview of the FapRΔ43–malonyl-CoA complex showing the main hydrogen-bonding interactions (in magenta) involved in the ‘zipper’ effect, which are absent in the ligand-free protein (see text). Loops A–C, which are disordered in ligand-free FapR, are shown in green. Download figure Download PowerPoint A model of action for FapR The malonyl-CoA-induced structural rearrangements described above severely constrain the spatial mobility of the linker helix αL with respect to the TLD core, supporting a mechanism for the regulation of transcription (Figure 7). According to this model, the flexibility of loop A in the repressed state allows the two DBDs of FapR to adopt a competent operator-binding conformation. When malonyl-CoA binds to FapR, a disorder-to-order transition of loop A during formation of the ligand-binding tunnel modifies the orientation of helix αL, pulling apart the DBDs from each other and disrupting their productive association for operator binding. This allosteric mechanism differs from that of other bacterial repressors, for which the ability to bind DNA typically involves a moderate hinge-bending motion between domains (Friedman et al, 1995; Orth et al, 2000; van Aalten et al, 2001). Figure 7.Proposed model of action of FapR. The figure shows the repressed (operator-bound, left) and derepressed (malonyl-CoA-bound, right) states of FapR. In each case, molecular surfaces represent the experimental X-ray structures. The DBDs (gray cylinders) are modeled on the MecI/BlaI repressor family (Garcia-Castellanos et al, 2003). The proposed αL–αL interaction for the repressed state is indeed observed between neighboring dimers in the crystal structure of the FapRΔ43–malonyl-CoA complex. Download figure Download PowerPoint Several lines of evidence support the above model. First, most residues involved in the ‘zipper’ effect are largely conserved in FapR proteins from different species (Schujman et al, 2003). Second, the structure of the complex shows the linker helices (αL) protruding away from the dimeric core in opposite directions (Figure 4), incompatible with an architecture allowing DNA-binding through the two N-terminal domains. In agreement with this observation, residues 68–73 from loop A, which are determinant for αL stiffening in the derepressed state, are present but disordered in two different crystal forms of unbound FapR, suggesting a flexible conformation. Such flexibility is confirmed by proteolysis experiments showing that loop A is accessible to trypsin in the ligand-free protein as well as in the presence of inactive acyl-CoA molecules. Instead,
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