The FadR·DNA Complex

TRANSCRIPTIONAL CONTROL OF FATTY ACID METABOLISM IN ESCHERICHIA COLI*

Yibin XuDagger , Richard J. Heath§, Zhenmei LiDagger , Charles O. Rock§, and Stephen W. WhiteDagger ||

From the Departments of Dagger  Structural Biology and § Biochemistry, St Jude Children's Research Hospital, Memphis, Tennessee 38105 and the  Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, January 9, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Escherichia coli, the expression of fatty acid metabolic genes is controlled by the transcription factor, FadR. The affinity of FadR for DNA is controlled by long chain acyl-CoA molecules, which bind to the protein and modulate gene expression. The crystal structure of FadR reveals a two domain dimeric molecule where the N-terminal domains bind DNA, and the C-terminal domains bind acyl-CoA. The DNA binding domain has a winged-helix motif, and the C-terminal domain resembles the sensor domain of the Tet repressor. The FadR·DNA complex reveals how the protein interacts with DNA and specifically recognizes a palindromic sequence. Structural and functional similarities to the Tet repressor and the BmrR transcription factors suggest how the binding of the acyl-CoA effector molecule to the C-terminal domain may affect the DNA binding affinity of the N-terminal domain. We suggest that the binding of acyl-CoA disrupts a buried network of charged and polar residues in the C-terminal domain, and the resulting conformational change is transmitted to the N-terminal domain via a domain-spanning alpha -helix.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fatty acid synthesis and degradation are important facets of bacterial physiology, and the regulation of these pathways has been extensively studied in the model prokaryote, Escherichia coli (1). Fatty acids are vital constituents of the cell membranes, but they also represent a source of energy. Thus, fatty acid degradative and biosynthetic metabolic pathways must be switched on and off based on the availability of extracellular fatty acids. In E. coli, the transcription factor, FadR, functions as a switch that coordinately regulates the machinery required for fatty acid beta -oxidation and the expression of a key enzyme in fatty acid biosynthesis (2, 3).

It has been known for some time that the enzymes responsible for fatty acid degradation in E. coli are inducible, and the isolation of regulatory mutants, fadR, suggested the existence of a single repressor that controlled the entire set of degradative (fad) genes (4-6). Mutagenesis studies identified the fadR gene and verified that this single repressor controls the transcription of the whole fad regulon (7, 8). Following the cloning (9) and sequencing (10) of the fadR gene, it was predicted that FadR contains a HTH1 motif, which is consistent with its proposed function as a DNA-binding protein. The fad genes are only induced in the presence of long-chain fatty acids, suggesting that FadR is a classical bacterial repressor. Thus, in the absence of fatty acids, the protein binds at a site downstream of the promoters of the fad genes and represses transcription. When long chain fatty acids become available, they are converted to CoA thioesters, bind to FadR, and elicit a conformational change that releases the protein from DNA, thereby removing the repression. Subsequent studies on the fadB gene (11) confirmed this general outline and revealed that the FadR DNA binding site in fadB is close to the +1 region relative to the start site of transcription.

The subsequent observation that fadR mutants were also defective in unsaturated fatty acid production (12) led to the important discovery that FadR is also a transcriptional activator (13, 14). Specifically, it controls the expression of the fabA gene that encodes the enzyme FabA, which introduces double bonds into the growing acyl chain (13, 14). Analysis of the fabA gene revealed a canonical FadR binding site in the -40 region of the promoter where positive activators of the sigma 70 factor typically bind. Thus, the role of FadR as a repressor or an activator depends on the location of its binding site within the promoter. When bound downstream of the RNA polymerase binding site of the fad promoters, it blocks the polymerase and acts as a repressor. However, when bound close to the -40 region, it promotes the binding of the RNA polymerase. A number of FadR binding sites have now been characterized, and all conform to this straightforward model (15).

To understand the mechanism of FadR at the molecular level, we have performed a crystallographic analysis of the protein. Here we report the structure of the FadR dimer at 1.5 Å in the absence of bound DNA and long chain acyl-CoA thioesters, and also the structure of the FadR·DNA complex at 3.25 Å. The crystal structure of FadR in the absence of DNA has recently been determined independently at 2.0 Å resolution (16), and therefore the emphasis of this report is how FadR interacts with DNA. Structural homologies to other transcription factors provide important clues as to how ligand binding controls the DNA affinity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning the fadR Gene-- The fadR open reading frame (GenBankTM/EBI accession X08087) was amplified from E. coli strain UB1005 in a polymerase chain reaction using the primers fadr-Nco (5'-CCATGGTCATTAAGGCGCAAAGC) and fadr-Bam (5'-GGATCCTTATCGCCCCTGAATGGC), and was cloned into pCR2.1 (Invitrogen). Following transformation into INValpha F' (Invitrogen), a plasmid with the correct sequence was isolated, and the gene was released by digestion with NcoI and BamHI. This fragment was ligated into similarly digested pET-15b (Novagen) to create pPJ139 for expression of full-length FadR. Plasmids were transformed into B834(DE3) (Novagen) for expression. To produce FadR containing selenomethionine ([SeMet]FadR), the cells were resuspended in minimal medium containing selenomethionine (0.05 g/liter; Sigma) immediately prior to induction. This method is fully described elsewhere (17). Generally, the level of FadR expression was greater than 20% of the soluble cell protein.

Purification of FadR-- Native FadR was purified by tandem anion and cation ion-exchange chromatography essentially as described previously (11). [SeMet]FadR did not bind to cation-exchange resins, and the following alternative purification method was developed. Cells from 1 liter of culture were resuspended in buffer A (20 mM Tris, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol) and lysed in a French pressure cell at 16,000 psi. Cell debris was removed by centrifugation, and the supernatant was directly applied to a 30 ml DE52 column pre-equilibrated with 20 mM Tris, pH 8.0. The protein was eluted with a linear gradient of KCl in 20 mM Tris, pH 8.0. The fractions containing FadR were identified by SDS-polyacrylamide gel electrophoresis and pooled. The pooled fractions were concentrated with an Amicon stirred cell and applied to a Sephacryl S200 gel filtration column equilibrated in buffer A containing 100 mM NaCl. FadR eluted as a single peak at a position consistent with a dimer and was essentially pure as judged by SDS-polyacrylamide gels.

Crystallization-- FadR in the absence of bound DNA was crystallized using the hanging drop procedure at 18 °C by mixing equal volumes of the protein solution at 10 mg/ml with a reservoir solution containing 0.1 M sodium citrate, pH 5.6, 0.7 M ammonium sulfate, 0.2 M lithium sulfate, and 5 mM zinc chloride. Although crystals grew from ammonium sulfate or lithium sulfate alone, a mixture of the two gave the best crystals. [SeMet]FadR crystallized in the same condition, but without zinc chloride. Crystals typically grew within 2 weeks and were in space group P21 with unit cell dimensions a = 59.4 Å, b = 87.0 Å, c = 59.1 Å, and beta  = 120.2°. There is one dimer in the asymmetric unit. To prepare the FadR·DNA complex, the oligonucleotides, 5'-CGATCTGGTCCGACCAGATGCT-3' and 5'-GCATCTGGTCGGACCAGATCGA-3', were dissolved in buffer A, annealed, and mixed with FadR at a protein/DNA molar ratio of 1:3. Crystals of the complex were also obtained by the hanging drop method at 18 °C by mixing equal volumes of 8 mg/ml complex solution with a reservoir solution containing 10% isopropyl alcohol, 50 mM MES, pH 6.0, and 10 mM MgCl2. Crystals grew in 1 month and were in space group P212121 with the cell dimensions a = 83.7 Å, b = 110.1 Å, and c = 130.2 Å. There is one dimer complex in the asymmetric unit.

Data Collection and Processing-- All diffraction data used in these studies were collected at the Structural Biology Center (SBC) beamline 19ID at the Advanced Photon Source in Argonne National Laboratory, Chicago. Data were recorded on a 3 × 3 CCD detector from crystals flash-frozen in liquid nitrogen at 100 K. MAD data were collected from Se-Met-labeled crystals that were cryoprotected with 30% sucrose. The peak, inflection point and high energy remote wavelengths were determined from an x-ray fluorescence spectrum collected from the mounted crystal, and 360° of data were collected at each wavelength in two 180° sweeps. The 1.5 Å native dataset was collected from an unlabeled frozen crystal that was also cryoprotected in 30% sucrose. Data were collected from a frozen FadR·DNA complex crystal that was cryoprotected in 20% MPD. Integration, scaling, and merging of data were performed with the program HKL2000 (18).

Structure Determination and Refinement-- The FadR structure in the absence of DNA was determined directly from the MAD data. All Patterson search, MAD phasing, electron density map calculations, and density modification procedures were carried out using the CNS program suite (19). Four selenium atoms were identified at equivalent locations in the unit cell from each of the three MAD datasets. These initial positions were refined and then used in a MAD phasing calculation at 1.7 Å resolution. The experimental electron density map was of high quality, but density modification produced a greatly improved and easily interpretable map at 1.7 Å resolution. A model was built into the density using the O program (20), and refined against the MAD remote data using simulated-annealing, positional, and individual B-factor refinement. The partially refined model was then further refined in the same manner against the superior quality 1.5 Å resolution native data. The final model includes residues 5-230 in each monomer, 416 water molecules, 6 sulfate ions, and 1 zinc ion located on the local molecular 2-fold axis. 95.5% of residues are in the core regions of a Ramachandran plot performed in PROCHECK (21), and only the two glutamine residues at position 6 in the dimer are in forbidden regions.

The structure of the FadR·DNA complex was solved with molecular replacement methods using AMoRe (22). The 1.5 Å resolution native FadR dimer was used as a search model. After rigid body refinement performed in AMoRe, the R-factor was 45.5% with a correlation coefficient of 0.505 calculated with data between 10.0 and 3.2 Å resolution. A preliminary model of the bound DNA molecule was built into the visible density, and the structure was improved iteratively with successive rounds of model building using the O program (20) and refinement using CNS (19). Only grouped B-factor refinement was applied. Non-crystallographic symmetry restraints were applied during the early stages of refinement and later removed. The final model contains one FadR dimer (residues 7 to 228), one DNA duplex, and one magnesium ion.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The FadR Crystal Structure-- The MAD technique, using metabolically incorporated selenomethionine, was used to solve the FadR structure. The selenium positions were identified using automated Patterson search methods, and phasing was carried out to the resolution limit of the MAD data (1.7 Å). The data collection and phasing statistics are shown in Table I. The electron density map after density modification was of superb quality, and the complete FadR molecule was visible apart from four residues at the N terminus and nine residues at the C terminus. Refinement by simulated annealing was performed against a native dataset that extended to 1.5 Å resolution. The refinement statistics and model quality parameters are listed in Table II.

                              
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Table I
Crystallographic data and phasing statistics for the FadR structure analysis

                              
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Table II
Refinement Statistics

The structure of the FadR monomer is shown in Fig. 1, and it is essentially identical to that reported previously (16). Briefly, it can be divided into three regions, an alpha /beta N-terminal domain (alpha 1-beta 1-alpha 2-alpha 3-beta 2-beta 3), an alpha -helical C-terminal domain (alpha 6-alpha 7-alpha 8-alpha 9-alpha 10-alpha 11-alpha 12), and a linker comprising two short alpha -helices (alpha 4-alpha 5). The two major domains only interact via the linker region, and the overall shape of the monomer therefore resembles a dumbbell. The structure of the N-terminal domain conforms to the so-called winged-helix motif (23), consistent with its role in binding DNA. The larger C-terminal domain is essentially an antiparallel array of six alpha -helices that form a barrel-like structure, with a seventh alpha -helix (alpha 10) forming a lid at the end closest to the N-terminal domain. FadR forms a dimeric structure in the crystal, which is consistent with independent studies showing a dimer to be the functional state of the protein in solution (24). The monomers are packed together in a parallel fashion, and the two domains and the linker regions make reciprocal interactions across the interface. The monomers are tightly wrapped, and ~1600 Å2 of surface area are buried in forming the dimer. A region of tetrahedral electron density on the local 2-fold axis was interpreted as a zinc atom coordinated to two water molecules and the Odelta 1 atoms of asparagines 81 and 81' (the prime refers to the second monomer in the dimer structure). Although zinc ions were not required to grow crystals of FadR, they were essential to obtain the highest resolution crystals from which the 1.5 Å native dataset was collected.


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Fig. 1.   The structure of the FadR monomer (left), and its homology to the winged-helix domain of CAP and the ligand binding domain of the Tet repressor (right). The monomer is color-coded to emphasize the proposed homologies: magenta, the winged-helix motif; green, the Tet repressor motif; red, the two additional helical regions in the C-terminal domain of FadR compared with the Tet repressor. The figure was produced using MOLSCRIPT (36) and rendered with Raster3D (37).

Homologous Structures to FadR-- Known protein structures with homology to FadR were identified using the Dali search engine (25). The coordinates of the N- and C-terminal domains were submitted separately. As noted earlier, the N-terminal domain shows clear homology to the winged-helix motif (23), and the Dali search identified CAP (26) as the most similar winged-helix transcription factor. A number of proteins were shown to have similar helical arrangements to the FadR C-terminal domain within their structures, for example ATP synthase, myosin, and serum albumin, but the most intriguing was the C-terminal domain of the tetracycline (Tet) repressor (27). Like FadR, the Tet repressor contains a regulatory C-terminal domain that recognizes a specific ligand (tetracycline), which, in turn, mediates its ability to bind cognate DNA (28). Fig. 1 shows the FadR, CAP, and Tet repressor domains in equivalent orientations to highlight their similarities.

Based on sequence alignment, it was previously suggested that FadR might be homologous to the GntR family of transcription factors (16, 29). However, the sequence homology is marginal, and there are no structures of any of the GntR family members to confirm this suggested relationship. We consider that the homologies to CAP and the Tet repressor based on structure are more relevant. Therefore, in terms of structural genomics, FadR can be categorized as a chimera of two motifs that have previously been characterized, the winged-helix motif and the C-terminal domain of the Tet repressor.

The Crystal Structure of the FadR·DNA Complex-- The structure of the FadR·DNA complex was determined by molecular replacement using the dimer as the search model. Phases from the molecular replacement solution produced an electron density map in which the sugar-phosphate backbone of the bound DNA was clearly visible. Iterative rounds of model building and refinement generated the final structure. Because the FadR·DNA complex is the asymmetric unit in the crystal, non-crystallographic symmetry constraints were incorporated into the early rounds of refinement, but these were relaxed in the final rounds. Also, to guard against model bias, a number of simulated annealing omit maps were calculated during refinement in which regions at the protein-DNA interface were removed from the model. A representative region of the final electron density map is shown in Fig. 2A, and the pertinent statistics of the final model are listed in Table II.


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Fig. 2.   The structure of the FadR·DNA complex. A, the 2Fo-Fc calculated phased map from the final refined coordinates of the FadR·DNA complex at 3.25 Å. The area shown is the recognition of G7, G8, and A16' by Arg-35, Arg-45, and His-65, respectively. The map is contoured at the 1.5 sigma  level and displayed using the O program (20). B, stereoview of the FadR·DNA complex. The central paired alpha 3 helices are in the major groove, and the two wings are within the flanking minor grooves. Note that the DNA is bent toward the FadR molecule, and that the major and minor grooves are contracted and expanded respectively as a result. The 5'-cytosine at one end of the DNA is flipped out of the duplex and makes crystal contacts with a neighboring FadR molecule. The picture was produced using the Ribbons program (38).

The complete structure of the FadR·DNA complex is shown in Fig. 2B. The conformation of the FadR dimer in the complex is virtually identical with that in the absence of bound DNA. The RMSD on alpha carbons is 0.717 Å. All of the duplex DNA is visible in the electron density map apart from the overhanging ends. In the crystal, the ends of the DNA interact with the C-terminal domains of neighboring FadR molecules, and the 5'-cytosine of one chain is flipped out of the duplex to form one of these interactions (Fig. 2B). Apart from the central G(11)-C(11') base pair, the DNA in the complex is palindromic, and the pseudo 2-fold axis is coincident with the local 2-fold axis of FadR, with the major groove facing the protein and the minor groove facing away from the protein. The result is that each N-terminal domain interacts in an identical fashion with its DNA half-site. The DNA has a B-form conformation with a curvature of 20° toward the protein, and this results in a contraction of the central major groove and an expansion of the opposite minor groove. The gross features of the B-form helix are recognized by the two N termini of the paired alpha 3 helices that project orthogonally into the central major groove and the two beta -ribbon wings that dock into the flanking minor grooves.

A detailed view of the FadR-DNA interface is shown in Fig. 3A, and only one monomer is presented, because both half-sites are identical. The interactions that determine the specificity are shown diagrammatically in Fig. 3B. Although there are a number of nonspecific interactions with the DNA sugar-phosphate backbone, only three adjacent base pairs are formally recognized within the complex, T/A (6/16'), G/C (7/15') and G/C (8/14'). These interact with His-65, and arginines 35 and 45 respectively, which are invariant in the three known sequences of FadR (16). His-65 is at the very tip of the wing and is buried deep in the minor groove where the Nepsilon 2 nitrogen forms a hydrogen bond with N3 of A16. The guanidinium groups of arginines 35 and 45 are hydrogen-bonded to the O6 and N7 atoms of G7 and G8 in the major groove. Other conserved residues in the complex include Arg-49, that forms a salt bridge interaction with the phosphate group of G8, and threonines 44, 46, and 47 at the N terminus of helix alpha 3 that interact with the sugar-phosphate backbone. Finally, glutamic acids 34 and 50' appear to be key residues, because they form electrostatic interactions with, and presumably stabilize the positions of, arginines 35, 45, and 49. 


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Fig. 3.   Details of the FadR·DNA complex showing specific protein-DNA interactions. A, stereoview showing a close up of the FadR-DNA interface within one monomer. See text for details. The picture was produced using the Ribbons program (38). B, schematic of the FadR-DNA interface showing the important residues and how they interact with the DNA. Amino acids from one monomer are shown in the same colored text. Note that the interactions are identical across the local 2-fold axis of the DNA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Architecture and Specificity of the FadR-DNA Interaction-- The structure of the FadR·DNA complex provides a firm basis for understanding how this transcription factor controls the expression of bacterial fatty acid metabolic genes. A key finding is that the N-terminal winged-helix motif does not bind DNA in the classical manner. The structures of several protein-DNA complexes involving winged-helix motifs have now been determined (23) and, typically, the second recognition helix in the HTH region binds along the DNA major groove, and the beta -ribbon binds within the adjacent minor groove. However, the precise details of the interaction can vary (23, 30), and FadR is unusual because only the N terminus of the alpha 3 recognition helix is within the major groove. The most similar example to the FadR variation is found in CAP (26), although, unlike CAP, FadR does not induce a sharp 90° bend in the DNA. It is therefore not surprising that the FadR and CAP winged-helix domains are structurally very similar. The way in which FadR binds DNA is determined by the location of the alpha 3 recognition helices that are paired together at the dimer interface. The FadR dimer cannot bind DNA with the alpha 3 helices along the major groove unless the N-terminal domains move apart to expose the surfaces of these helices. This was implied in a model of the complex based on the apo-FadR structure (16), but we can now confirm that the N-terminal domains continue to interact via their alpha 3 helices in the FadR·DNA complex.

The oligonucleotide that was used to produce crystals of the FadR·DNA complex was based on the highest affinity cognate sequence that is found within the fadB promoter (11). The structure reveals that only three residues (Arg-35, Arg-45, and His-65) formally recognize this sequence, and FadR appears to have specificity for the palindromic consensus sequence 5'-TGGNNNNNCCA-3'. It is clear why the two G/C base pairs in each half-site are important specificity elements in the complex (Fig. 3). Both are involved in specific and identical Arg/G/C triple interactions, and the adjacent triples are able to form a stable stacked configuration within the half-site. The specificity for the flanking T/A base pairs in the minor groove is probably related to the close approach of the wing and the sidechain of His-65, which is facilitated by the lack of a sidechain on the adjacent and conserved Gly-66. A G/C base pair in this position would introduce an amine group into the minor groove (N2 from the guanine), and the close approach would be compromised. Finally, the five central spacer elements are necessary to match the architecture of the dimeric N-terminal domains and the resulting distances between the 2-fold related specificity elements. The consensus sequence based on our structure is supported by binding data on other cognate sequences where deviations generally reduce the affinity (15). However, the conservation within these sequences also suggests that a better consensus is 5'-TGGTNNNACCA-3', and this is consistent with independent in vitro selection studies on the FadR binding site within the iclR promoter (31). The inner T/A base pair is present in the oligonucleotide used in this study, and a specific interaction is feasible with Arg-49 that is adjacent in the complex. However, this is not supported by our structure in which Arg-49 forms a salt bridge with Glu-50'.

The FadR Switching Mechanism-- Studies both in vitro and in vivo have revealed that long-chain acyl-CoA thioesters are the actual ligands that control the DNA binding affinity of FadR, and that they interact directly and reversibly with a specific region of the C-terminal domain (14, 24, 32-34). Based on the functionally and structurally related Tet repressor that has been fully characterized by crystallographic studies (27), we can predict that the interaction results in a conformational change that affects the structure, and hence the DNA binding affinity, of the N-terminal domain. In the Tet repressor, ligand binding within the C-terminal domain causes the movement of an alpha -helix that connects to, and also forms part of, the N-terminal domain (27). In the paired N-terminal domains, the result of this movement is that the two DNA binding helices are no longer able to bind in the major groove. Therefore, the connecting alpha -helix essentially represents a mechanism for communicating structural changes from the regulatory domain to the DNA binding domain. By analogy, to establish how the FadR switching mechanism operates, it is necessary to understand the nature of the ligand-induced conformational change, the interdomain communication mechanism, and the resulting effects on the N-terminal domain.

Mutagenesis experiments have identified Gly-216, Glu-218, Ser-219, Trp-223, and Lys-228 within helix alpha 12 as components of the acyl-CoA binding site. Also, affinity labeling experiments with a palmitoyl-CoA analog tagged the peptide 187-195 in the adjacent helix alpha 11 (32). The interior of the C-terminal domain contains an unusual cluster of aromatic, charged, and polar residues, which is directly adjacent to these putative acyl-CoA binding regions. The cluster is composed of tyrosines 172, 193, and 215, Arg-105, Thr-106, Asp-145, and Ser-219, and it is knitted together by a lattice of hydrogen-bonding interactions that involves five buried water molecules (Fig. 4A). This lattice links together five of the seven surrounding alpha -helices including alpha 11 and alpha 12, and we suggest the binding of acyl-CoA disrupts this lattice and produces a large conformational change within the C-terminal domain. This suggestion is based on structural studies of the BmrR transcription factor, which controls the expression of the Bmr multidrug-efflux transporter in Bacillus subtilis (35). The C-terminal sensor domain of BmrR contains a similar buried cluster composed of three tyrosines and a glutamic acid, which forms part of the binding site for aromatic/hydrophobic cationic drugs. The structure of the drug-bound complex showed that the aromatic ligand accesses this buried array and induces a large conformational change in the sensor domain.


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Fig. 4.   The FadR switch mechanism. A, stereoview of the putative switch region in the C-terminal domain of FadR. This constellation of buried aromatic, polar, and charged residues is extensively hydrogen-bonded into a tight lattice and adjacent to the putative acyl-CoA binding site involving helices alpha 11 and alpha 12. Note that the lattice includes a number of water molecules, and it directly links five of the seven alpha -helices that comprise the C-terminal domain. For clarity, the two remaining helices, alpha 7 and alpha 9, are not shown in the figure. B, structure of the FadR dimer color-coded to emphasize the important interfaces that we suggest are important to the switching mechanism. Note the polar residues on helix alpha 3 at the interface of the N-terminal domains and the aromatic and hydrophobic residues at the boundaries of the N- and C-terminal domains. The green sphere is the putative zinc ion at the local 2-fold axis. The orange circle indicates the location in the C-terminal domain of the constellation shown in A. The figure was produced using MOLSCRIPT (36) and rendered with Raster3D (37).

As regards the interdomain communication mechanism, helix alpha 5 in the linker region appears to be a key component, because it has extensive and conserved hydrophobic interfaces with the two flanking domains (Fig. 4B). Specifically, leucines 86 and 89, and Ile-82 interact with the N-terminal domain, and leucines 80 and 83, and Ala-87 interact with the C-terminal domain. The interface with the N-terminal domain is also characterized by a conserved cluster of aromatic residues comprising tryptophans 21, 60, and 75, and Phe-74. Therefore, helix alpha 5 may have a similar role to the equivalent domain-spanning helix of the Tet repressor. Unfortunately, the BmrR structural studies do not provide clues as to how the conformational change in the sensor domain modulates DNA affinity because the DNA binding domain is missing (35). One scenario for FadR is that the interface between the alpha 3 helices is broken by movements in the C-terminal domain, and this leads to a disruption of the precise DNA binding architecture of the N-terminal domain dimer. This is plausible because the interface between the N-terminal domains is only mediated by the paired alpha 3 helices, and the interactions are exclusively ionic and polar (Fig. 4B). The 2-fold related arrays of interacting amino acids comprise arginines 49, 54', and 57, Glu-50', Gln-53, and Asp-58'. Glu-13 from helix alpha 1 is the only other residue within FadR that contributes to this interface. Thus, a separation of the N-terminal domains would not be structurally unfavorable, because it would only result in the exposure of two hydrophilic surfaces.

    ACKNOWLEDGEMENTS

We are particularly grateful to Rongguang Zhang and the rest of the SBC staff at the APS for help in collecting and processing the diffraction data. We also thank Hee-Won Park for invaluable help, and Suzanne Jackowski and Allen Price for insights and discussions. Pam Jackson, Amy Sullivan, Xiaoping He, and Charles Ross II provided excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM34496 (to C. O. R.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities (ALSAC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1HW1 and 1HW2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

|| To whom correspondence should be addressed: Dept. of Structural Biology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3040; Fax: 901-495-3032; E-mail: stephen.white@stjude.org.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100195200

    ABBREVIATIONS

The abbreviations used are: HTH, helix-turn-helix; CAP, catabolite gene activator protein; CoA, coenzyme A; FabA, beta -hydroxydecanoyl-ACP dehydratase; MAD, multiwavelength anomalous dispersion; MPD, 2-methyl-2,4-pentanediol; RMSD, root mean-squared deviation; MES, 4-morpholineethanesulfonic acid; Tet, tetracycline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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