The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR

Abstract

FadR is an acyl-CoA-responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The apo-protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl-COA: The recently described structure of apo-FadR shows a DNA binding domain coupled to an acyl-CoA binding domain with a novel fold, but does not explain how binding of the acyl-CoA effector molecule > 30 A away from the DNA binding site affects transcriptional regulation. Here, we describe the structures of the FadR-operator and FadR- myristoyl-CoA binary complexes. The FadR-DNA complex reveals a novel winged helix-turn-helix protein-DNA interaction, involving sequence-specific contacts from the wing to the minor groove. Binding of acyl-CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5 A. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 A in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl-CoA-responsive regulation.

Full text

Daan M.F.van Aalten
1
, Concetta C.DiRusso
2
and Jens Knudsen
3
Wellcome Trust Biocentre, Division of Biological Chemistry and
Molecular Microbiology, School of Life Sciences, University of
Dundee, Dow Street, Dundee DD1 5EH, UK,
2
The Center for
Cardiovascular Sciences, Albany Medical College, Albany, NY,
USA and
3
Department of Biochemistry and Molecular Biology,
Odense University, DK-5320 Odense M, Denmark
1
Corresponding author
e-mail: dava@davapc1.bioch.dundee.ac.uk
FadR is an acyl-CoA-responsive transcription factor,
regulating fatty acid biosynthetic and degradation
genes in Escherichia coli. The apo-protein binds DNA
as a homodimer, an interaction that is disrupted by
binding of acyl-CoA. The recently described structure
of apo-FadR shows a DNA binding domain coupled to
an acyl-CoA binding domain with a novel fold, but
does not explain how binding of the acyl-CoA effector
molecule >30 A
Ê
away from the DNA binding site
affects transcriptional regulation. Here, we describe
the structures of the FadR±operator and FadR±
myristoyl-CoA binary complexes. The FadR±DNA
complex reveals a novel winged helix±turn±helix
protein±DNA interaction, involving sequence-speci®c
contacts from the wing to the minor groove. Binding
of acyl-CoA results in dramatic conformational
changes throughout the protein, with backbone shifts
up to 4.5 A
Ê
. The net effect is a rearrangement of the
DNA binding domains in the dimer, resulting in a
change of 7.2 A
Ê
in separation of the DNA recognition
helices and the loss of DNA binding, revealing the
molecular basis of acyl-CoA-responsive regulation.
Keywords: acyl-CoA/fatty acid/protein structure/
regulation/transcription
Introduction
The anabolic and catabolic pathways of fatty acid
metabolism in Escherichia coli consist of enzymes that
are signi®cantly different from their mammalian counter-
parts (DiRusso et al., 1999). The E.coli fatty acid synthase
system is of type II, consisting of individual dissociated
enzymes (Rock and Cronan, 1996), and has been a
successful target for the development of antibiotics such as
triclosan and thiolactomysin (Jackowski et al., 1989;
Waller et al., 1998; Levy et al., 1999). Degradation of fatty
acids occurs through a multifunctional enzyme complex
(DiRusso et al., 1999) coupled to a unique fatty acid
uptake pathway, including an active membrane transporter
(FadL) and an acyl-CoA synthetase (FadD), allowing the
bacterium to grow on long-chain fatty acids as a sole
energy source (DiRusso et al., 1999). To allow adaptation
to various stages in the cell cycle and concentrations of
fatty acids in the media, these anabolic and catabolic
routes need to be balanced through precise transcriptional
regulation. This is achieved by the acyl-CoA-responsive
transcription factor FadR (DiRusso et al., 1999). In the
absence of long-chain acyl-CoAs, FadR directly binds to
speci®c DNA sequences (DiRusso et al., 1999) and acts as
a repressor for transcription of genes involved in fatty acid
degradation (fadE, fadF, fadG and fadBA) and import
(fadD and fadL). Simultaneously, transcription of biosyn-
thetic genes (fabA and fabB) is activated. The products of
these genes, b-hydroxydecanoyl-acyl carrier protein
dehydrase and b-ketoacyl-acyl carrier protein synthase II,
respectively, play a crucial role in the synthesis of
unsaturated long-chain fatty acids (Rock and Cronan,
1996). Apart from binding DNA, FadR is able to bind
long-chain acyl-CoAs directly with a K
d
of 50±400 nM
(Raman and DiRusso, 1995). Binding of acyl-CoA is
presumed to result in a conformational change and leads to
inhibition of DNA binding (DiRusso et al., 1992, 1993).
This loss of DNA binding then results in derepression of
the catabolic genes, and deactivation of the anabolic
genes. The exact mechanism and structural details of
effector binding have been the subject of speculation
(DiRusso et al., 1999), but so far have not been studied
experimentally.
Recently, the structure of FadR has been determined in
the absence of both DNA and acyl-CoA (van Aalten et al.,
2000a). This study revealed an unusual overall fold, not
previously observed in currently known regulator struc-
tures. The N-terminal DNA binding domain is of the
winged helix±turn±helix (wHTH) type, and the C-terminal
domain contains a new fold consisting of a seven-helical
bundle with cross-over topology. This bundle was sub-
sequently shown to contain a large cavity (142 A
Ê
3
), lined
by several residues that were shown, by mutagenesis
studies, to be involved in acyl-CoA binding (Raman and
DiRusso, 1995). This cavity, located >30 A
Ê
away from the
DNA binding site, was thus suggested to be the putative
site of binding of the effector molecule, long-chain
acyl-CoA. Although this putative site of binding was
identi®ed, it remained unclear what were (i) the structural
details of the FadR±DNA interaction, (ii) the mode of
binding of the effector and (iii) the nature and mechanism
of effector-induced conformational changes leading to
disruption of the FadR±DNA complex.
Here, we describe the crystal structures of FadR in
complex with an operator DNA duplex at 3.25 A
Ê
reso-
lution, and of FadR bound to an effector, myristoyl-CoA,
at 2.1 A
Ê
resolution, which de®ne the FadR±DNA inter-
action, the site and mode of effector binding, and
demonstrate the presence of large effector-induced con-
formational changes leading to disruption of the protein±
DNA complex.
The structural basis of acyl coenzyme A-dependent
regulation of the transcription factor FadR
The EMBO Journal Vol. 20 No. 8 pp. 2041±2050, 2001
ã European Molecular Biology Organization
2041

Results and discussion
Overall structure of the FadR±operator complex
The FadR±operator complex was solved by molecular
replacement (aided by the anomalous signal from an
incorporated gold atom) and re®ned to 3.25 A
Ê
resolution
(Table I). The structure (Figure 1A) reveals dimeric FadR
bound to a DNA duplex, corresponding to the natural fadB
operator, on which FadR acts as a transcriptional
repressor. Each FadR monomer binds to a semi-sym-
metrical half of the operator through its wHTH motif.
Apart from interacting with the DNA (total buried surface
1892 A
Ê
2
), there are also interactions between the DNA
binding domains themselves (buried surface 266 A
Ê
2
). The
orientation of the DNA binding domains with respect to
the effector binding domains is similar to the apo-structure
[root mean square deviation (r.m.s.d.) on all C
a
atoms in
the dimer = 0.8 A
Ê
], suggesting that the apo-protein is able
to bind DNA, which is expected as the acyl-CoA- and
DNA-bound states are mutually exclusive. The structure
of the operator appears to be affected by binding to the
protein. The central 3 bp are bulged out, as evidenced by a
translation of ~5 A
Ê
of the helical axis compared with the
axis de®ned by the base pairs at the ends of the 19meric
operator. This is further supported by a roll (7° and 9°,
away from the minor groove) of the two base-pair steps
neighbouring the central base pair, and a signi®cant
widening of the minor groove (to 9.1 A
Ê
) opposite
the central base pairs. Overall, however, the DNA
helix corresponds to canonical B-DNA, with proper
Watson±Crick hydrogen bonding for all 19 bp, and no
intercalated protein side chains.
Details of the FadR±DNA interaction
The contacts between FadR and the DNA helix are
summarized in Figure 1B and shown in Figure 1C. All
contacts are symmetrical, with the exception of the Arg35
interaction with a guanine (Figure 1B). FadR side chains
interacting with the DNA, and the DNA helix itself, were
well de®ned in the electron density maps (Figure 1C). No
water-mediated protein±DNA contacts were observed,
although this may be a function of the relatively low
resolution of the diffraction data. Five different areas of
each FadR monomer interact with the DNA. Residues 7±9,
just prior to the start of helix a1 in the wHTH motif, make
several non-speci®c contacts with the phosphate back-
bone, possibly providing support to the more speci®c
contacts downstream. Mutation of Ala9 to valine disrupts
the ability of FadR to interact with DNA (Raman et al.,
1997). The start of helix a2 (the ®rst helix in the classical
HTH motif) provides a contact between the backbone of
Glu34 and the phosphate backbone, and contains Arg35,
which donates two speci®c hydrogen bonds to acceptor
atoms on the major groove side of guanine at the ±4
position (Figure 1B and C). Mutation of Arg35 abrogated
the FadR±DNA interaction, as shown by alanine-scanning
mutagenesis and random hydroxylamine mutagenesis
(which generated the Arg35Cys mutant) (Raman et al.,
1997). The third area of interaction constitutes the turn in
the HTH motif. The side chain of Thr44 hydrogen bonds to
the phosphate backbone and provides a hydrophobic
contact to the cytosine/guanine in the central base pair.
Arg45 donates hydrogen bonds to the guanine at the ±3
position, comparable to the Arg35±guanine interaction
(Figure 1C). The fourth and ®fth areas of FadR±DNA
contacts are somewhat different from previously observed
wHTH±DNA interactions (Gajiwala and Burley, 2000).
Helix a3, normally termed the recognition helix, contacts
the major groove using residues on the ®rst helical turn
only. Thr46 provides speci®c contacts with the central G-C
base pair, and Thr47 donates a hydrogen bond to a nearby
backbone phosphate group. The last residue on helix a3 to
interact with the DNA helix is Arg49, which donates two
hydrogen bonds to the phosphate backbone. The Arg49Ala
mutation was shown to inhibit DNA binding completely
(Raman et al., 1997). The last FadR±DNA contact area
constitutes residues 63±69, which correspond to wing W1
in the standard wHTH nomenclature (Gajiwala and
Burley, 2000) as they form the loop between b-strands
b2 and b3, which, together with b1, form a small b-sheet.
This loop deeply invades the minor groove, with the C
a
atom of Gly66 lying approximately on an axis connecting
the ring oxygens of the ribose units forming the minor
groove (Figure 1A and C). Thr69 hydrogen bonds to the
phosphate backbones, whereas Lys67 points between two
phosphates on the backbone, but does not approach them
closely enough (4.3±6.2 A
Ê
) to form proper hydrogen
bonds. It is possible that there is still a small electrostatic
contribution to the binding from this residue, as evidenced
from mutagenesis studies which showed a small loss of
binding for the Lys67Ala mutant, as qualitatively assessed
by gel shift assays (Raman et al., 1997). An unusual
protein±DNA contact is formed by His65, located at the tip
of the wing, which extends even further into the minor
groove. The imidazole ring is located in the minor groove
Table I. Details of data collection and re®nement
FadR±myristoyl-
CoA
FadR±operator
Space group P6
1
22 P6
1
22
Unit cell (A
Ê
) a = 59.48 a = 93.00
b = 59.48 b = 93.00
c = 290.48 c = 334.83
Resolution, last bin (A
Ê
) 30±2.1
(2.18±2.1)
25±3.25
(3.37±3.25)
Unique re¯ections 18 556 14 138
Redundancy 4.7 (4.9) 6.2 (4.5)
Completeness (%) 97.8 (95.5) 98.0 (98.6)
R
merge
0.038 (0.292) 0.143 (0.458)
I/sI 17.9 (5.1) 6.6 (2.5)
R
cryst
0.226 0.271
R
free
0.256 0.309
No. of atoms 1781 protein 3631 protein +
2 Au + 3 Cl
134 water 28 water
63 ligand 773 DNA
Wilson B-factor (A
Ê
2
) 39.4 n.d.
<B> protein (A
Ê
2
) 42.5 67.8
<B> DNA (A
Ê
2
) ± 61.8
<B> (adenosine + pyrophosphate) 65.9 ±
<B> (pantetheine + myristate) 48.8 ±
<B> (water) 50.5 35.2
R.m.s.ds from ideal geometry
bond lengths (A
Ê
) 0.009 0.013
bond angles (°) 1.46 1.7
main chain B-factors (A
Ê
2
) 1.7 1.1
Data were collected at 100 K (l = 0.92 A
Ê
for FadR±myristoyl-CoA
and l = 1.025 A
Ê
for the FadR±operator complex). All measured data
were included in the re®nement.
D.M.F.van Aalten, C.C.DiRusso and J.Knudsen
2042

bordering base pairs 4/5. His65 hydrogen bonds with
guanine at ±4 and adenine at +5, through its Ne2 atom. In
addition (or alternatively, depending on the protonation
state of this histidine), Nd2 atom forms a hydrogen bond
with the oxygen in the ribose ring at guanine +6 (Figure 1B
and C). Substitution of His65 and Gly66 for other amino
acid residues inhibits DNA binding completely (Raman
et al., 1997).
The FadR±DNA contacts that may determine sequence
speci®city are found on the central C-G base pair (0), the
two guanines at ±3 and ±4, and the adenine at +5. It has
been shown that FadR protects its fadB operator from
treatment with dimethylsulfonate at guanines 0, ±3 and ±4
(the latter two on both strands) (C.C.DiRusso and
N.Raman, unpublished results), which is in agreement
with our structural data. The DNase I footprint of FadR
was determined to be 17 bp (DiRusso et al., 1992). In our
structure, the contact farthest away from the central
binding site is the Lys67 electrostatic interaction with the
backbone phosphate connecting base pairs ±7 and ±8,
which would thus agree approximately with the measured
footprint. Although fadB is the strongest known binding
site for FadR [K
d
= 0.2 nM (DiRusso et al., 1992)], seven
other fad binding sites are known, with the consensus
5¢-NRCTGGT(A/C)YGAY(G/C)(T/A)N(T/A)N-3¢ (N,
any; R, purine; Y, pyrimidine). Although it is worth
noting that this consensus is not symmetrical (see also
Figure 1B), the two guanines that interact tightly with
Arg35 and Arg45 on FadR are conserved.
FadR is a member of the wHTH family, the topology of
which normally consists of a1±b1±a2±turn±a3±b2±W1±
b3±W2, where W denotes a `wing' (Lai et al., 1993).
Recent structures have emphasized the diverse ways in
which these domains can interact with DNA. Helix a3,
termed the recognition helix, normally occupies the major
groove de®ning the larger part of the speci®city of the
protein±DNA interaction (Gajiwala and Burley, 2000). In
the recently published hRFX1 structure, however, a new
mode of DNA binding was observed, with the recognition
helix contacting the minor groove, whereas W1 occupied
the major groove, making the most of speci®c contacts
(Gajiwala et al., 2000). The structure of a heat shock
transcription factor DNA binding domain complexed with
DNA revealed another orientation, with weak recognition
helix±DNA contacts and a wing primarily involved in
protein±protein interactions (Little®eld and Nelson, 1999).
While describing the structure of apo-FadR (van Aalten
et al., 2000a), we proposed a model of the FadR±DNA
interaction that was based on the structure of the E2F-
DP±DNA complex (Zheng et al., 1999). The approximate
areas of interaction agree between the proposed model and
our present structure. However, compared with the
FadR±DNA complex described here, the recognition
helix (and the DNA binding domain) in the model was
rotated too far towards the DNA helix. E2F4 binds DNA
with its recognition helix pushed into the major groove,
and with its single wing barely touching the outside of the
phosphate backbone. Here, we have shown that the
FadR±operator complex represents an unusual wHTH
protein±DNA interaction, in which only the tip of the
recognition helix a3 is involved in speci®city contacts
and wing W1 penetrates the minor groove of the helix,
where it can make speci®c contacts with two bases
(Figure 1B and C).
FadR is a member of the GntR family of prokaryotic
transcription factors, for which no structures were
available before the determination of the apo-FadR crystal
structure (van Aalten et al., 2000a). We have aligned a
representative set of sequences from this family to FadR
and evaluated sequence conservation in the context of the
present FadR±operator complex structure (Figure 2). The
®rst 10 residues of FadR are not part of the GntR signature,
which basically consists of the secondary structure
elements from helix a1 to the last strand b3. Glu34
appears to be conserved, and although its side chain does
not interact with DNA, it seems to serve to keep Arg45 and
Arg49 together (Figure 1C). Arg35, involved in speci®c
contacts, occurs in other GntR sequences, but is not well
conserved. Two other important arginines, Arg45 (speci®c
contacts) and Arg49 (phosphate backbone contacts), are
both conserved. The threonines interacting with the central
base pair (Thr44, Thr46 and Thr47) are highly variable in
the GntR family, but completely conserved in the FadRs.
Although His65, which interacts with the minor groove
(Figure 1B and C), is not found in other GntR sequences,
Gly66 is highly conserved. Inspection of the FadR±DNA
complex (Figure 1C) reveals the reason for this: the Gly66
backbone is located deep within the minor groove, and any
side chain atoms incorporated at this position would
introduce steric clashes with the DNA. Taken together, it
seems likely that the overall GntR±DNA interactions will
be similar to the FadR±operator complex.
Binding of myristoyl-CoA to FadR
The structure of FadR complexed with myristoyl-CoA was
solved by molecular replacement and re®ned to 2.1 A
Ê
resolution. The electron density for the effector was
located in the C-terminal acyl-CoA binding domain
(Figure 3A), as predicted from the native structure
(van Aalten et al., 2000a). The adenosine 3¢-phosphate
moiety is partially solvent exposed and covers some of the
more hydrophobic atoms on the pantothenic acid moiety,
as seen in many other acyl-CoA binding proteins (Engel
and Wierenga, 1996; Lerche et al., 1997; van Aalten et al.,
2001). The pantothenic acid moiety penetrates the seven-
helix bundle through a channel between helices a5 and
a10, such that the acyl chain is buried deep inside the
acyl-CoA binding domain (Figure 3A). Three main areas
of the ligand show interactions with the protein
(Figure 3B). The adenosine 3¢-phosphate and pyrophos-
phate units form several hydrogen bonds (some water
mediated) with solvent-exposed protein residues, most
notably Arg213 (conserved in other FadRs; Figure 2),
whose positively charged side chain is well positioned to
interact with the negative charges on the pyrophosphate.
Polar atoms on the pantothenic acid moiety form hydrogen
bonds to residues on the protein (Thr106 and Ser219,
conserved in other FadRs; Figure 2). The thioester oxygen
accepts a hydrogen bond from Arg105 and the C
14
acyl
chain is buried in a pocket surrounded by hydrophobic
residues (Figure 3B). The acyl chain adopts a bent
conformation and terminates in a pocket mainly formed
by residues from helices a5 and a8 (Ile108, Ile164,
Leu165). The size of the pocket appears to leave room for
additional methylene groups. Long-chain acyl-CoAs have
Structure of the FadR±myristoyl-CoA complex
2043

D.M.F.van Aalten, C.C.DiRusso and J.Knudsen
2044

been shown to bind FadR, with K
d
s for binding of C
14:0
-,
C
16:0
- and C
18:1
-CoA of 59, 369 and 63 nM, respectively,
measured by isothermal titration calorimetry (DiRusso
et al., 1998). FadR is apparently able to bind acyl-CoAs
containing an acyl chain longer than C
14:0
, with high
af®nity, in agreement with the extra space observed in the
present structure.
Several amino acid substitutions have been reported that
cause a FadR `super-repressor' phenotype, i.e. rendering
FadR unable to dissociate from DNA in the presence
of long-chain acyl-CoAs. These substitutions, namely
Tyr179®Ala, Gly216®Ala, Ser219®Asn and Trp223®
Ala, were suggested to lie at positions lining the acyl-CoA
binding site (Raman and DiRusso, 1995), although they
could also affect the conformational changes necessary to
transduce the signal to the DNA binding domains. Analysis
of the location of these residues with respect to
myristoyl-CoA bound to FadR shows that they all line
the acyl-CoA binding pocket (Figure 3B) and that most of
them are conserved in the sequence of other FadRs
(Figure 2). Tyr179 changes conformation upon acyl-CoA
binding, lying tightly packed against the acyl chain, and
substitution to alanine would disrupt this interaction.
Trp223 has only weak direct contacts with the ligand,
but may act as a `seal' at the bottom of the hydrophobic
binding pocket, protecting it from the solvent. Mutation to
alanine would disrupt the contacts with the ligand and
would expose the binding pocket to the solvent (Figure 3B).
Fluorescence studies have con®rmed a role of Ser219 in
ligand binding (DiRusso et al., 1998), and it is worth noting
that this is one of the few residues conserved in the effector
binding domain (Figure 2). Introduction of any side chain
atom beyond C
a
on Gly216 or a larger side chain instead of
Ser219 would change the tight interactions these residues
have with the pantothenic acid moiety into severe steric
clashes. Thus, our structural data are in agreement with
available experimental data and point to several other key
residues whose mutation could have signi®cant effects on
acyl-CoA binding (Figure 3B).
Effector-induced conformational changes
As pointed out earlier, the protein in the apo-FadR and
FadR±DNA complexes is in a similar conformation. The
effector binding domains are slightly more similar, and
superimpose with an r.m.s.d. of 0.57 A
Ê
on the C
a
atoms.
Given the higher resolution data for the apo-FadR
structure (2.0 A
Ê
, compared with 3.25 A
Ê
for the
FadR±operator structure), here we study the ®ner details
of the acyl-CoA-induced conformational changes by
comparing the apo-FadR and myristoyl-CoA±FadR struc-
tures, and subsequently discuss the large conformational
changes by comparison of the FadR±operator and
FadR±myristoyl-CoA complexes. Although myristoyl-
CoA appears to ®t into a pocket within the acyl-CoA
binding domain in the apo-FadR and FadR±operator
stuctures, this is not possible without signi®cant con-
formational changes. Prior to effector binding, Met168 and
Tyr172 partially occupy the pocket, as shown in Figure 4A.
Upon binding of myristoyl-CoA, these residues undergo
dramatic conformational changes. Met168 is forced to
rotate 92° around c
2
and ends up pointing towards the
outside of the helical bundle. Tyr172 rotates 44° around
c
2
, but even in that position has many close contacts with
the methylene groups of the acyl chain (Figure 4A). While
Tyr172 is conserved in other FadRs (Figure 2), Met168 is
less conserved and a leucine also appears possible at that
position. This may re¯ect a tuning of speci®city towards
length or saturation of the fatty acid. To further relieve
Fig. 1. (A) Structure of the FadR±DNA complex. The FadR±DNA complex is shown in two orientations (along and perpendicular to the pseudo 2-fold
axis). The DNA binding domains in the FadR homodimer are coloured green and blue, the effector binding domains are shown in black. The tips of
the wing in the wHTH motif are coloured magenta and the DNA duplex is shown as a red sticks model. (B) Schematic overview of FadR±DNA
contacts. The 19 bp DNA duplex is shown with purines represented by long green bars and pyrimidines by short purple bars. The rounded boxes
represent side chains on the protein interacting with the DNA (blue, monomer A; red, monomer B), with interactions shown by black arrows.
(C) Stereo image of the DNA binding domains bound to DNA. C
a
traces of the DNA binding domains in the dimer are shown in grey and blue. For
the blue domain, the wing and the loop in the wHTH motif are coloured green, and side chains contacting the DNA (de®ned as contact distance less
than the sum of van der Waals radii + 0.5 A
Ê
) are shown in sticks representation, with carbons coloured black. Ten base pairs of the DNA duplex are
shown as sticks, with the ®nal 2F
o
± F
c
electron density map contoured at 1.25 s (magenta). Hydrogen bonds between FadR and DNA are shown as
dotted black lines, and can also be identi®ed in Figure 1B. Hydrogen bonds originating from FadR backbone nitrogens are drawn as originating from
the corresponding C
a
atom.
Structure of the FadR±myristoyl-CoA complex
2045

their steric clashes with the bound effector, these two
`sensor' residues induce conformational changes in the
backbone (C
a
positional shifts of up to 3.5 A
Ê
; Figure 4A).
Helix a8 is already bent in the apo-FadR and
FadR±operator structures, and almost forms two separate
helices. In the structure of the complex with myristoyl-
CoA, the effector-induced conformational changes in the
sensor residues kink helix a8, in effect forming two
separate helices (Figure 4A). As a result of the kink in
helix a8, some of the side chains on this helix are pushed
towards the neighbouring helix a4, which responds by
shifting away from helix a8 towards the N-terminal DNA
binding domain (Figure 4A). This shift of helix a4
introduces additional contacts with helix a1 in the
N-terminal domain, which tilts away, taking the entire
DNA binding domain with it in a rigid body motion
(Figure 4A). Thus, it appears that binding of
myristoyl-CoA in the effector binding domain, >30 A
Ê
away from the site of DNA binding, causes a hinge-
bending motion of the entire DNA binding domain over an
angle of 13°, resulting in backbone shifts of up to 4.5 A
Ê
.
Apart from the large rigid body movements described
above, the effector binding domain does not show other
large shifts. When the effector binding domains of the
FadR±myristoyl-CoA and apo-FadR structures, excluding
helices a4 and a8, are superposed, this results in an
r.m.s.d. of 0.3 A
Ê
on all atoms. Perhaps this is not
surprising, as it was already possible to detect nearly the
entire ligand binding pocket in the apo structure
(van Aalten et al., 2000a). The largest side chain
conformational change in the pocket, apart from Met168
and Tyr172, is Arg105, which rotates around c
2
±c
4
to
form a hydrogen bond with the thioester oxygen
(Figure 3B). This residue is conserved in other FadR
sequences (Figure 2).
We have reported previously on diffraction data of a
crystal grown from solutions containing FadR and
octanoyl-CoA (van Aalten et al., 2000b). The unit cell
dimensions and space group of this crystal form
(a = b = 59.7 A
Ê
, c = 296.2 A
Ê
, P6
1
22) are similar to
those presented here for the FadR±myristoyl-CoA
structure (Table I). Interestingly, a partially re®ned struc-
ture from a gold derivative of this crystal form (which
yielded the highest resolution data: 2.6 A
Ê
, R = 0.265,
R
free
= 32.9) did not reveal ordered ligand density, and the
two `sensor' residues Met168 and Tyr172 were in the same
conformation as in the apo-FadR structure. However, the
protein adopts an overall conformation similar to the
myristoyl-CoA structure, although the conformational
changes are not as large (superpositions on C
a
s:
apo-FadR ± FadR±myristoyl-CoA = 2.1 A
Ê
; apo-FadR ±
FadR±octanoyl-CoA = 1.8 A
Ê
), so this could be considered
an `in-between' structure. This agrees with the biochem-
ical data, which indicate that octanoyl-CoA may bind
weakly to FadR, but is not able to inhibit DNA binding. It
may be that a highly disordered, non-interpretable ligand
is present in the octanoyl-CoA crystals, causing the
conformational change, although a C
8
acyl chain is too
short to reach both sensor residues (Figure 3B). Another
possibility is that apo-FadR is able to populate the ligand-
bound conformation in con®gurational space, which is
then preferred in the P6
1
22 space group.
Acyl-CoA-induced domain motions
Having de®ned structures of apoFadR (van Aalten
et al., 2000a) and the FadR±operator and
FadR±effector complexes, it is now possible to under-
stand the effect of acyl-CoA binding in the context of
a FadR dimer interacting with DNA. As noted above,
binding of myristoyl-CoA leads to a displacement of
Fig. 2. Sequence alignment of E.coli FadR (FADR_ECOLI) and putative FadRs from Haemophilus in¯uenzae (FADR_HAEIN) and Vibrio cholerae
(FADR_VBCHO), together with a representative group of signature sequences from the GntR family, indicated by their standard SwissProt accession
codes. FadR sequence numbering is used, and the secondary structure elements are indicated by bars and arrows. Conserved residues are indicated by
black boxes. Residues touching the acyl-CoA ligand or the DNA helix are indicated by black circles.
D.M.F.van Aalten, C.C.DiRusso and J.Knudsen
2046

the entire DNA binding domain with respect to the
effector binding domain. The function of this motion
becomes clear upon inspection of the conformational
change by comparing the FadR±myristoyl-CoA and
FadR±operator complexes (Figure 4B). Because of the
(pseudo) 2-fold axis relating the two monomers in the
homodimer, the effector-induced hinge bending of the
DNA binding domains occurs in opposite directions.
This leads to a variation of the distance between the
two recognition helices from 8 A
Ê
(FadR±operator
complex; 7 A
Ê
in the apo-FadR structure) to 14 A
Ê
(FadR±myristoyl-CoA), and shifts the tip of the wing
in each DNA binding domain by up to 4 A
Ê
.
Thus, binding of the effector molecule, in this case
myristoyl-CoA, controls the separation between the
recognition helices, and thus the ability to interact with
the DNA helix. This mechanism is similar to that for
the tetracycline repressor (Orth et al., 2000) and the
Fig. 3. (A) Stereo image of FadR±myristoyl-CoA structure and ligand electron density. A C
a
trace is shown, coloured according to secondary
structure. Helices are coloured red (DNA binding domain) or blue (acyl-CoA binding domain), strands green and turns grey. Secondary structure
elements are labelled. The ligand, myristoyl-CoA, is shown as a stick model with carbons coloured black. A simulated annealing F
o
± F
c
map is also
shown, contoured around the ligand at 2.25 s (green). (B) Stereo image of protein±ligand interactions. The protein backbone is shown as a blue trace.
Side chains touching the ligand (contact distance less than the sum of van der Waals radii + 0.5 A
Ê
) are shown as sticks coloured by atom type.
Myristoyl-CoA is shown as a stick model with carbons coloured black. Water molecules interacting with the ligand are shown as green spheres.
Hydrogen bonding with the ligand is indicated by black dashed lines.
Structure of the FadR±myristoyl-CoA complex
2047

purine repressor (Friedman et al., 1995), where the
ability to bind DNA is lost by an opening of the DNA
binding domains.
Concluding remarks
The structures of the FadR±myristoyl-CoA and FadR±
operator complexes described here have revealed the
Fig. 4. (A) Stereo image of the superposition of a monomer from the apo-FadR structure onto the FadR±myristoyl-CoA complex. For areas showing
relatively little conformational change, only the backbone trace of the monomer from the apo-FadR structure is shown in grey. Helices a4 and a8 in
the acyl-CoA binding domain and the entire DNA binding domain are show in blue for apo-FadR, and in magenta for the FadR±myristoyl-CoA
complex. Side chains of residues Met168 and Tyr172 are shown with blue carbons for apo-FadR and magenta carbons for the FadR±myristoyl-CoA
complex. Myristoyl-CoA is shown as a stick model, with carbons coloured black. (B) Stereo image of the superposition of the FadR±operator complex
onto the dimer of the FadR±myristoyl-CoA complex. The FadR±operator structure is coloured grey and FadR±myristoyl-CoA is coloured blue, with
red DNA-recognition helices. Residues Met168 and Tyr172 are shown with carbons coloured orange (FadR±operator) or magenta (FadR±myristoyl-
CoA). Myristoyl-CoA is shown as a stick model, with carbons coloured black. The DNA helix is shown as a thin black sticks model.
D.M.F.van Aalten, C.C.DiRusso and J.Knudsen
2048

details of the FadR±effector and FadR±DNA interaction,
and the nature and extent of effector-induced conform-
ational changes. The data show that effector binding
results in signi®cant conformational changes transmitted
from the effector binding domain to the DNA binding
domain, leading to a conformational state that is no longer
favourable for interaction with DNA. Although many
structures of effector binding domains complexed to their
corresponding effectors have been described (e.g. Egea
et al., 2000), not many examples are known of structures
of full-length transcriptional regulators bound to their
effectors. One of the most well characterized systems is
the catabolite gene activator protein (CAP) family
(Schultz et al., 1991), with the most recent example
being the structure of CooA (Chan, 2000; Lanzilotta et al.,
2000). These proteins are similar to FadR to the extent that
they also bind DNA as homodimers, and that regulation of
DNA binding is dependent on the binding of an effector
molecule. However, whereas CAP regulators acquire the
ability to bind DNA upon binding of their effectors, the
FadR±DNA interaction is inhibited by effector binding.
Interestingly, it has been shown that for the lactose
repressor family (LacI) and the purine repressor (PurR),
such opposite functions can exist within the same struc-
tural framework (Schumacher et al., 1994, 1995; Friedman
et al., 1995; Bell and Lewis, 2000). Binding of their
respective effector molecules increases the af®nity for
DNA in the case of PurR, but disrupts the LacI±DNA
interaction. Although the effector-bound structures are
similar, the PurR±corepressor complex binds DNA,
whereas the LacI±inducer complex does not. In a manner
analogous to FadR, the transcription factors from the LacI
family appear to regulate DNA binding by separation of
their N-terminal, HTH motif-containing DNA binding
domains.
The transcription factor most similar to FadR from a
structural and mechanistic point of view is the tetracycline
repressor, TetR. Native TetR binds tightly to DNA through
the helix±turn±helix motifs in the N-terminal domains of
the homodimer (Orth et al., 2000). Binding of tetracycline
at the interface of the all-helical C-terminal domains
induces conformational changes of side chains in the
binding pocket, which are then translated into shifts of
a-helices, ultimately resulting in the two DNA binding
domains moving farther apart, leading to dissociation from
the DNA. The chain of events upon effector binding in
FadR is similar, also leading to an opening of the DNA
binding domains.
The GntR family of prokaryotic transcription factors
regulate pathways as diverse as gluconate synthesis (Miwa
and Fujita, 1988), trehalose metabolism (Schoeck and
Dahl, 1996) and glycolate oxidation (Pellicer et al., 1996).
Many of these transcription factors show sequence
homology to FadR's DNA binding domain (Figure 2),
and secondary structure predictions suggest that some
members of the GntR family also possess seven-helical
effector binding domains (van Aalten et al., 2000a). This
implies that FadR might serve as a structural scaffold for
this new family of regulators. Since the FadR±effector and
FadR±effector complexes have de®ned the details of
interaction with effector and DNA, it may be feasible in
the future to predict these interactions for other GntR
regulators.
The de®nition of the effector binding site in FadR, and
determination of the conformational state of the protein
bound to DNA, enable studies towards inhibitory mol-
ecules that would block the ligand binding site. This could
open up possibilities for interfering with the normal
transcription of fatty acid biosynthetic genes, which have
been shown to be an excellent target for antibiotics
(Jackowski et al., 1989; Waller et al., 1998; Levy et al.,
1999).
Materials and methods
Puri®cation, crystallization and data collection
FadR was overexpressed in E.coli and puri®ed as described previously
(DiRusso et al., 1998). The resulting protein solution contained 4.6 mg/ml
FadR in 50 mM KH
2
PO
4
, 10% glycerol pH 8.0. A 2-fold molar excess of
myristoyl-CoA was added prior to crystallization. Hanging drop vapour
diffusion crystallization experiments were set up using 1 ml of solution
containing the complex and an equal volume of well solution containing
20% PEG 8000, 200 mM magnesium acetate and 100 mM sodium
cacodylate pH 6.5. Crystals were grown at 20°C, and reached a size of
~0.2 3 0.2 3 0.2 mm within 2±3 weeks. They were transferred into
mother liquor containing 10% ethylene glycol, and after 60 s frozen in a
nitrogen gas stream (100 K) for data collection.
For the FadR±operator complex, a 1.1-fold molar excess was added of
a DNA duplex consisting of 19meric oligos 5¢-CATCTGGTA-
CGACCAGATC-3¢ and 5¢-GATCTGGTCGTACCAGATG-3¢, which
were HPLC puri®ed prior to annealing. Hanging drop vapour diffusion
crystallization experiments were set up using 1 ml of solution containing
the complex and an equal volume of well solution containing 1.6 M
sodium citrate pH 6.5. Small bipyramidal needles grew at 20°C, and
reached a size of ~0.1 3 0.05 3 0.05 mm within 2 weeks. The presence
of DNA in the crystals was con®rmed by ethidium bromide staining and
mass spectrometry. A crystal was transferred into mother liquor
containing 10 mM AuCN for 30 min, and then transferred to a
cryoprotecting solution of mother liquor containing 5% glycerol. After
60 s, the crystal was frozen in a nitrogen gas stream (100 K) for data
collection.
Data were collected on a MAR CCD detector at beamline X11, at the
EMBL outstation at the DESY synchrotron, Hamburg (FadR±myristoyl-
CoA), and on an ADSC-Q4 detector at beamline F2 at CHESS, Cornell
University, using a wavelength (l = 1.025 A
Ê
) optimized for a gold
anomalous signal (FadR±operator). Images were processed using
DENZO and re¯ections merged using SCALEPACK of the HKL-suite
(Otwinowski and Minor, 1997). Data collection and processing statistics
are summarized in Table I.
Structure determination and re®nement
The FadR±DNA structure was solved by molecular replacement with
AMoRe (Navaza, 1994) using the native structure of the FadR dimer
[Protein Data Bank (PDB) entry 1E2X] as a search model. The
correctness of the solution was veri®ed by calculating a map from the
measured anomalous signal combined with the model phases, which
showed 6s peaks for gold atoms near the single solvent-exposed cysteine
in both monomers, which had previously been exploited in phasing the
native structure (van Aalten et al., 2000a). These cysteines are far away
from the DNA binding site, and it has been shown that reaction with the
heavy atom does not result in signi®cant conformational changes
(van Aalten et al., 2000a). After rigid body re®nement in CNS
(Bru
È
nger et al., 1998), in which the DNA binding and effector binding
domains were treated as separate rigid bodies, the R-factor was 0.421
(R
free
= 0.494, 25±3.25 A
Ê
data). The resulting maps showed density for
the DNA duplex, which was built in several steps using O (Jones et al.,
1991), starting with the central 5 bp. Further re®nement with CNS and
REFMAC included non-crystallographic symmetry restraints across the
pseudo 2-fold axis relating the monomers in the homodimer. Progress was
monitored by inspection of maps calculated from the anomalous signal,
which veri®ed the strength and location of the gold atoms. After
completion of the DNA model, the R-factor had fallen to 0.315
(R
free
= 0.361) with 25±3.25 A
Ê
data. After further macrocycles that
included adjustment of the protein model, placement of a few ordered
water molecules and partially occupied gold sites, the R-factor was 0.264
(R
free
= 0.320). Statistics of the model are shown in Table I.
Structure of the FadR±myristoyl-CoA complex
2049

For the myristoyl-CoA±FadR complex, initial molecular replacement
calculations using AMoRe (Navaza, 1994), with the native structure
(PDB entry 1E2X) as a search model, were unsuccessful, suggesting that
signi®cant structural rearrangements might have occurred upon binding
of myristoyl-CoA. Subsequent calculations in which only the C-terminal
acyl-CoA binding domain (Figure 1A) was used gave a single clear
solution after rigid body re®nement (R-factor 0.451, correlation
coef®cient 0.432, 8.0±4.0 A
Ê
data). An initial round of re®nement in
CNS (Bru
È
nger et al., 1998) resulted in maps in which the N-terminal
DNA binding domain could be positioned manually. Further rigid body
re®nement in CNS brought the R-factor down to 0.405 (R
free
= 44.5) with
25±3.75 A
Ê
data. Inspection of the maps revealed several regions of
disorder, which could not be interpreted unambiguously. The phases of
this partial solution were used as input for iterative re®nement and
autobuilding with warpNtrace (Perrakis et al., 1999). This resulted in a
model with 189 out of 240 possible residues, giving R = 0.322,
R
free
= 0.343 after simulated annealing in CNS. The maps had improved
and most of the missing regions and parts of the ligand could be seen in
the F
o
± F
c
difference maps. Iterative model building with O (Jones et al.,
1991) and re®nement with CNS (Bru
È
nger et al., 1998) resulted in a ®nal
model (R = 0.226, R
free
= 0.256) that included all atoms in the ligand
(Figure 1A) and residues 5±227 of the protein (Table I). The coordinates
and structure factors of both structures have been submitted to the PDB
(entries 1H9G and 1H9T).
Acknowledgements
We thank the EMBL-Hamburg outstation at the DESY synchrotron for
use of beamline X11 and the CHESS synchrotron at Cornell University
for their generous allocation of time on beamlines F1/F2. D.v.A. is
supported by a Wellcome Trust Career Development Research
Fellowship.
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Received December 15, 2000; revised January 24, 2001;
accepted February 27, 2001
D.M.F.van Aalten, C.C.DiRusso and J.Knudsen
2050