From the Departments of Biological Structure and
¶ Biochemistry, § Biomolecular Structure Center, and
** Howard Hughes Medical Institute, University of Washington, Seattle,
Washington 98195, and the
Department of Microbiology, University
of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, August 18, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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Iron-dependent regulators are primary
transcriptional regulators of virulence factors and iron scavenging
systems that are important for infection by several bacterial
pathogens. Here we present the 2.0-Å crystal structure of the wild
type iron-dependent regulator from Mycobacterium
tuberculosis in its fully active holorepressor conformation.
Clear, unbiased electron density for the Src homology domain 3-like
third domain, which is often invisible in structures of
iron-dependent regulators, was revealed by density modification and averaging. This domain is one of the rare examples of
Src homology domain 3-like folds in bacterial proteins, and, in
addition, displays a metal binding function by contributing two
ligands, one Glu and one Gln, to the pentacoordinated cobalt atom at
metal site 1. Both metal sites are fully occupied, and tightly bound
water molecules at metal site 1 ("Water 1") and metal site 2 ("Water 2") are identified unambiguously. The main chain carbonyl
of Leu4 makes an indirect interaction with the
cobalt atom at metal site 2 via Water 2, and the adjacent residue,
Val5, forms a rare Iron, because of its ability to participate in electron
transfer reactions with redox potentials ranging from +300 to Iron-dependent regulators
(IdeRs)1 are metal-activated
DNA-binding proteins found in Gram-positive and acid-fast bacteria. These proteins are transcriptional regulators of virulence factors as
well as siderophore synthesis enzymes and their associated transport
proteins (7, 8). If the intracellular iron concentration is
sufficiently high, transcription of virulence factors and iron import
pathways is repressed by the binding of Fe(II)-IdeR complexes to the
promoter/operator sequences for their genes (9, 10). The observation by
Pappenheimer et al. (11) that iron availability regulates
production of diphtheria toxin by Corynebacterium
diphtheriae led to the discovery of the first
iron-dependent regulator, the diphtheria toxin repressor
(DtxR) (12). DtxR, when in complex with Fe(II), represses transcription
of the tox gene of corynebacteriophage Initial crystal structures revealed DtxR (17, 18) to be a dimer with
three domains and two metal sites per monomer. Domain 1 (residues
1-74) is the DNA binding domain and contains a helix-turn-helix motif.
Domain 2 (residues 75-140) is the dimerization domain and contains
most of the metal binding residues. Domain 3 (residues 141-226)
appeared to have an SH3-like fold and to be connected to domain 2 by a
flexible linker (19). Mutational studies (20-22) have confirmed the
functional importance of residues involved in metal binding, and
structures have been solved that contained the transition metal ions
Fe(II), Co(II), Ni(II), Zn(II), Mn(II), or Cd(II), which have been
shown to act as corepressors of DtxR in vitro (23).
Structures of metal-bound DtxR in complex with DNA in three distinct
crystal forms (24-26) revealed that each 19-base pair
pseudo-palindromic operator sequence binds two dimers of DtxR. The
complex of Co(II)-DtxR-DNA by Pohl et al. (25) revealed
domain 3 in a new position in which it, quite surprisingly, contributes
two metal ligands to metal site 1, providing the first example of a
metal binding function for an SH3-like domain and implicating this
domain in the metal-dependent repressor activation mechanism.
The ideR gene from M. tuberculosis has been
cloned (27), and the IdeR protein, which shares ~88% sequence
identity to DtxR within the first 140 residues of this 230 residue
protein, has been shown to be a functional homologue of DtxR. It
regulates expression of Here we present the 2.0-Å crystal structure of wild type M. tuberculosis IdeR in complex with Co(II). This structure contains, for the first time, domain 3 of IdeR and reveals its SH3-like fold and
metal binding function. This is the most complete and highest
resolution structure of an iron-dependent regulator to date
in what we believe to be the fully active holorepressor conformation. Several features of the environments of the metal binding sites that
were hinted at by previous work but remained questionable have been
confirmed unambiguously. These features, which include several tightly
bound water molecules and precise ordering of the N-terminal residues
to form a Crystallization--
Wild type M. tuberculosis IdeR
was purified as described by Pohl et al. (30). For
crystallization the protein was concentrated to ~8-10 mg/ml (as
judged by the Bio-Rad protein assay) by evaporation in a Savant
SpeedVac on low temperature and dialyzed thoroughly at 4 °C against
a buffer containing only 50 mM NaCl, 10 mM
Tris-HCl, pH 7.0, using the microdialysis technique described by
Overall (31). This procedure was adopted because a significant amount of the protein was lost upon concentration with membrane concentrators, apparently due to adhesion to the membrane.
DNA used for crystallization was purchased from Macromolecular
Resources as the reverse-phase high performance liquid
chromatography-purified, single-stranded oligonucleotides. Duplex
oligonucleotides were formed by mixing equimolar volumes (as judged by
absorption spectroscopy at 260 and 280 nm) of complementary
oligonucleotide dissolved in 5 mM MgCl2, 50 mM Tris-HCl, pH 7.5, and annealing by fully submerging in
an 80 °C water bath, which was then allowed to cool gradually to
room temperature. The homogeneity of the annealed duplexes was judged
by capillary electrophoresis on a Bio-Rad BioFocus 3000. The
oligonucleotide used for crystallization of IdeR was a blunt-ended
21-base pair duplex formed by annealing oligonucleotides with the
sequences 5'-ATTAGGTTAGCCTAACCTAAA-3' and 3'-TAATCCAATCGGATTGGATTT-5'.
IdeR and annealed DNA duplex were mixed in an approximate 3:1 molar
ratio (~0.3 mM IdeR monomer and ~0.1 mM
DNA) with 5 mM CoCl2 and incubated on ice for
30 min prior to use. Crystallization was performed at room temperature
by hanging drop vapor diffusion by mixing equal volumes of
Co(II)-IdeR-DNA solution and precipitant solution on silanized
coverslips over wells containing 500 µl of precipitant solution.
Crystals were obtained from 2.0 M LiSO4, 0.01 M MgCl2, 0.05 M MES, pH 5.6, and
grew to a maximum size of ~500 × 300 × 150 µm within
1-2 weeks. Analysis of the diffraction pattern revealed the crystals
to be in space group P212121 with cell dimensions a = 46.30, b = 113.97, and c = 236.21 Å.
Data Collection--
Initial diffraction data were collected at
the Advanced Light Source (ALS) beamline 5.0.2 at Lawrence Berkeley
National Laboratory on a Quantum 4 CCD detector at a wavelength of 1.0 Å. The data were reduced with Mosflm (Andrew G. W. Leslie, MRC
Laboratory of Molecular Biology) and Scala (Phil Evans, MRC Laboratory
of Molecular Biology) and converted to structure factors with Truncate (32). A second diffraction data set was collected at the Advanced Photon Source (APS) beamline 19-ID at Argonne National Laboratory on
the APS1 3 × 3 CCD detector at a wavelength of 1.0332 Å. These data were reduced at APS with HKL2000 (33) and converted to structure
factors with Truncate. The crystals were flash-frozen in liquid
nitrogen after immersion for ~20 s in a cryoprotectant consisting of
a 20% weight/volume solution of dl-threitol in the crystal
mother liquor. Diffraction from these crystals was anisotropic. Analysis of the reduced intensities by the fall-off procedure (originally from the Groningen BIOMOL package) as implemented in
Truncate showed the crystals to diffract most strongly along the
crystallographic b axis and most weakly along the
crystallographic a axis. Data collection statistics are
presented in Table I.
Structure Determination--
The structure was solved by
molecular replacement in AMoRe (34) with the ALS data set from 20.0 Å to 3.5 Å, using as a search model the Zn(II)-IdeR structure (30). This
dimeric structure contains only the first two domains (residues 4-140)
of IdeR. AMoRe located two clear solutions. The first solution had an
initial correlation coefficient (CC) of 15.8 and R-factor of
54.8%, which was improved to a CC of 38.4 and R-factor of
50.9% after rigid body refinement. Addition of the second solution
gave a CC of 35.6 and R-factor of 48.5%, which improved to
a CC of 43.6 and R-factor of 46.8% after rigid body
refinement. These solutions represented the 21 helical
packing arrangement of IdeR along the crystallographic a
axis, with an ~40-Å wide solvent channel running through the center.
This two-domain solution provided no crystal contacts along the
b and c axes, suggesting that a significant portion of the model, presumably domain 3, was yet to be included.
The initial two-domain solution was refined briefly by the conjugate
direction method with TNT (35). Each domain (residues 4-74 and
75-140) was restrained by the 4-fold improper noncrystallographic symmetry. Clear and unbiased density for domain 3 was then revealed by
density modification and averaging; a combination of solvent flattening, histogram matching, and NCS averaging was performed with
DM (36) beginning with
Examination of
Rfree decreased steadily as the model building
and refinement progressed, and plots of log(
Solvent-accessible surface area calculations, superimpositions, and
other coordinate analyses were performed with EDPBD (42). Figures were
made with MOLSCRIPT (43), BOBSCRIPT (44), RASTER3D (45), and GRASP
(46).
Overall Structure--
The crystal structure of wild type M. tuberculosis IdeR in complex with Co(II) has been solved at 2.0-Å
resolution and refined to an R-factor of 21.4% with an
Rfree of 27.2% and good geometry (Table
I). The electron density is of excellent
quality and reveals nearly the complete polypeptide chain for four
25-kDa IdeR monomers (designated as A, B, C, and D) in the asymmetric
unit that form two functional dimers. As determined by PROCHECK (47),
99.2% of peptide The SH3-like Third Domain--
Clear and unbiased electron density
obtained by density modification and averaging, as described under
"Experimental Procedures," revealed domain 3 of IdeR for the first
time (Fig. 3). This domain, like domain 3 of DtxR determined by Qiu et al. (19) and Pohl et
al. (25), has an SH3-like fold with six
Domain 3 is positioned in the groove between domains 1 and 2 (Fig.
4) and contributes two ligands,
Glu172 and Gln175, to metal site 1. The
interactions of domains 3 with domains 1 bury 307-357 Å2
of solvent-accessible surface for domain 1 and 295-347 Å2
for domain 3. The interactions of domains 3 with domains 2 bury 537-656 Å2 for domain 2 and 532-566 Å2 for
domain 3. This interaction of domain 3 of IdeR with the other two
domains is essentially identical to that of the "wedge" position of
domain 3 of DtxR as observed in the Co(II)-DtxR-DNA complex (25).
As suggested by the range of buried surface areas reported above, the
position of domain 3 is slightly different in each IdeR monomer. The
C Metal Site 1--
The Co(II) atoms at metal sites 1 were
identified in
Additional peaks of 6.6, 6.7, 5.4, and 4.2 Metal Site 2 and the N-terminal Residues--
The Co(II) atoms at
metal sites 2 of the four IdeR monomers were identified as 23.8, 23.3, 11.5, and 9.3
In a number of previous structural determinations of wild type DtxR,
metal site 2 has been disrupted by an apparent oxidative modification
of Cys102 S
The sixth ligand to the Co(II) atom in metal site 2, which completes
the octahedral coordination, is a solvent molecule that is a water or
possibly a hydroxyl ion. This solvent appeared as 6.9, 6.6, 6.2, and
6.2
In our new structure of IdeR, in contrast to most previous structures
of iron-dependent regulators, the complete N terminus is
well ordered in all four monomers (Fig.
7) with excellent electron density (Figs.
2, 6). Met1 folds into the functional dimer interface,
making hydrophobic contacts with Val5 and the aliphatic
portion of Asp6 from its dimer mate. Residues 2-5 form a
type I hydrogen-bonded
It should be mentioned here that the unusual Additional Metal Binding Sites--
Examination of the
In monomers A, B, and C, two of the coordinating water molecules from
the metal site 3 Co(II) atom bridge it to a tightly bound sulfate ion
~5 Å away. These sulfate ions were identified as roughly tetrahedral
peaks of average 8.4
The importance of this newly identified metal site is unknown. Metal
binding at the corresponding site has not been reported for any DtxR
structure. This, together with the inconsistent binding interactions of
the associated sulfate ions, suggests that the occupation of metal site
3 is a result of crystallization in the presence of Co(II) and a high
concentration of lithium sulfate. Further investigation might prove otherwise.
Crystallographically related domains 3 from monomers B, C, and D form a
fourth Co(II) binding site (metal site 4) that is probably important
for the growth of this crystal form. The N Domain Orientation: Comparison to Apo-DtxR and
Co(II)-DtxR-DNA--
Qiu et al. (17) proposed that
activation of DtxR occurs by a reorientation of the DNA binding domains
by a metal binding induced hinge motion at about residue 74. This
hypothesis was upheld by the comparison of the structures of apo-DtxR
and Zn(II)-DtxR determined by Pohl et al. (54), which showed
a clear hinge bending motion in the predicted region. To further
investigate the hinge bending properties of iron-dependent
regulators, the relative domain orientations for this current
Co(II)-IdeR structure were examined by superimposing the C Role of DNA Oligonucleotides in Crystallization--
The
P212121 crystal form of IdeR was
obtained while attempting to crystallize a complex of IdeR with DNA.
The 21-base pair duplex DNA oligonucleotide present in the
crystallization trials was never visible in electron density maps, but
it seems to have been essential as an additive for the crystal growth
or nucleation. After the P212121
crystal form was discovered, controlled crystallization experiments
showed that these crystals could not be obtained unless the DNA was
included. Because all components of the crystallizations were carefully
buffered, the pH was not significantly altered by the presence of the
DNA. Therefore, we speculate that some interaction of DNA with IdeR is
required for crystallization. The only region in the crystal capable of
accommodating the DNA is an ~40-Å-wide solvent channel formed along
the a unit cell direction. The DNA-binding helices from
domains 1 of all monomers of IdeR in the unit cell project into this
solvent channel, suggesting that the DNA is involved in forming or
stabilizing the helical array of IdeR that forms this channel. However,
due to the close packing interactions between domains 1 of every
monomer, it would be impossible for the DNA to be bound in this crystal
form in the same manner as is observed for DNA bound to DtxR (24-26).
If DNA does exist in this solvent channel, it must be highly
disordered, for there were no features in the electron density maps
that could be reasonably interpreted as DNA in either the ALS or APS
data sets.
The 2.0-Å structure of IdeR from M. tuberculosis
represents another significant step toward an understanding of the
mechanism of activation of iron-dependent regulators. The
high resolution data revealed the first detailed picture of the metal
binding sites and N-terminal residues in the fully activated
conformation of an iron-dependent regulator, with the
benefit of four crystallographically independent observations. In
addition, the SH3-like fold and metal binding function of domain 3 were
confirmed by unbiased electron density. Because the ionic radii of
Co(II) and Fe(II) are very similar even in different coordination
states (55), and because Co(II) has been shown to be an excellent
corepressor of DtxR (23), we propose that the Co(II)-IdeR and
Fe(II)-IdeR structures are very similar and that domain 3 also displays
its metal binding function in vivo in the Fe(II)-IdeR complex.
At metal site 1 a buried water molecule, Water 1, was shown to
participate in a network of electrostatic interactions involving the
Glu83 and Gln175 ligands to the metal site 1 Co(II) atom and residues Arg80, Ser126, and
Asn130. Because of its pivotal position in bridging the
metal binding ligands of domain 3 with domain 2, Water 1 might have an
important role in metal-dependent activation of IdeR. Such
a role would be consistent with recent mutagenic experiments by
Goranson-Siekierke et al. (21), in which it was shown that
alanine substitution of either Arg80, Ser126,
or Asn130 disrupted repressor activity in C. diphtheriae DtxR even more severely than alanine substitution of
the actual metal liganding residues at metal site 1. Arg80
is particularly crucial as it bridges all three domains by making direct electrostatic interactions with the metal ligand
Glu172 from domain 3 and with Glu20 from domain
1. Ser126 and Asn130, however, interact with
the metal site 1 ligands Glu83 and Gln175 only
indirectly via Water 1.
Residues Arg80, Ser126, and Asn130
were previously postulated to form an anion binding site that allowed
orthophosphate to act as a co-corepressor in concert with the bound
metal ion (19). The Co(II)-DtxR-DNA (25) complex revealed that this
site is able to accommodate the metal binding ligands from domain 3, and this current high resolution Co(II)-IdeR complex has revealed the
participation of the tightly bound Water 1 and its potential importance
in the regulator's function.
At metal site 2, the S The Co(II)-water-Leu4 carbonyl interaction is part of a
network of electrostatic and hydrophobic interactions that stabilize the distinct conformation of the N-terminal residues, including the The N-terminal residues might serve to properly orient and stabilize
the DNA-binding helices of domain 1 to bind to DNA. In the current IdeR
structure, the conformation of the N-terminal residues leads to a
substantial interaction between domains 1 that buries an additional
~320 Å2 of solvent-accessible surface area at the dimer
interface. In most previous structures, the interaction between domains
1 was much less substantial and buried only ~87 Å2 of
accessible surface area. In the Ni-Cys102 The current Co(II)-IdeR structure provides an opportunity to further
evaluate the hinge bending properties of iron-dependent regulators. The relative domain orientations among the known structures are consistent with the previous proposals by Qiu et al.
(17) and Pohl et al. (54) of a hinge-binding motion at about
residue 74 being involved in metal-dependent activation.
The Co(II)-IdeR structure presented here is the first structure of a
non-DNA-bound regulator with the SH3-like domain in the "wedge"
position, the N-terminal residues well ordered, and both metal sites
fully occupied. It is surprising, therefore, that it does not show the
same orientation of the DNA-binding domains as do the Co(II)-DtxR-DNA
(25) and Ni(II)-Cys102 The variation in relative domain orientations among these structures
presents the possibility that the fully metal-activated holorepressor
conformation is not an exact fit to the DNA, but that a further
conformational change occurs upon binding to target DNA. In the known
complexes of DtxR with DNA (24-26), the DNA itself is notably
distorted, raising the intriguing possibility that the inherent
flexibility of the DtxR target sequences is a crucial aspect of
recognition. This hypothesis is inspired by recent work by Lee et
al. (57), which has shown that substitutions of nucleotides in
DtxR-specific operators that are not contacted by DtxR in the crystal
structures can have dramatic effects on DtxR binding and promoter
strength. Additionally, studies of the effect of noncontacted bases on
DNA recognition by the 434 repressor (58) and P22 repressor (59)
support the importance of "indirect readout" in recognition of DNA
by proteins (60). Further biochemical experimentation and structural
studies will be required to fully reveal the importance of
conformational flexibility of both iron-dependent
promoter/operator sequences and iron-dependent regulators.
turn. Residues 1-3 are well ordered
and make numerous interactions. These ordered solvent molecules and the conformation and interactions of the N-terminal pentapeptide thus might
be important in metal-dependent activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
500 mV
(1), is a virtually indispensable cofactor in important biological
processes, including respiration, photosynthesis, nitrogen fixation,
and DNA synthesis. However, despite its natural abundance, the
biological availability of iron is extremely low. In neutral solution
ferrous iron, Fe(II), rapidly oxidizes to ferric iron, Fe(III), and
forms insoluble hydroxides (KS ~ 10
18 M). Iron is often a limiting
factor for growth of living organisms, and in the case of pathogenic
organisms it is a limiting factor in infection (2). Higher organisms
such as humans sequester iron in tight complexes with iron storage and
transport proteins such as transferrin, lactoferrin, and ferritin,
making the iron unavailable to invading bacteria (3). To obtain
sufficient iron from their environment, many bacteria secrete low
molecular weight chelators called siderophores, such as the exochelins
and mycobactins of Mycobacterium tuberculosis (4).
Ferric-siderophore complexes are actively imported by membrane-spanning
receptor proteins. Pathogens also secrete proteins capable of
extracting iron from heme and iron transport proteins of the host (3, 5), and iron-dependent production of virulence factors has been observed in many bacteria (6).
, which encodes
diphtheria toxin, by binding to a target sequence within the
tox operator (8, 12). Seven other DtxR-specific iron-regulated promoter/operators (IRPs), designated IRP1-IRP6 (13-15) and hmuO (16), have also been reported.
-galactosidase under control of the
tox, IRP1, or IRP2 DtxR-specific promoter/operators in
response to high and low iron conditions (28). It has also been shown
to regulate transcription of fxbA, a gene needed for
exochelin synthesis in Mycobacterium smegmatis (29). The
2.6-Å structure of M. tuberculosis IdeR (30) revealed the
first two domains and showed both metal sites fully occupied, but the
intriguing third domain, which shares only ~24% sequence identity
with DtxR, was invisible.
turn centered at Val5, may be important in
metal-dependent activation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A-weighted (37) observed
structure factors phased on the partially refined model. NCS averaged
electron density maps were then calculated with FFT (Lyn F. Ten Eyck)
and Maprot (38). These maps allowed residues 151-207 and 216-228 (~30% of the total protein mass of IdeR) to be built de
novo. This model for domain 3 packed perfectly into the unit cell
to provide the missing contacts along the b and c
axes of the unit cell.
A-weighted Fobs
Fcalc maps with Peakmax (Phil Evans) allowed
placement of 13 apparent Co(II) atoms in the asymmetric unit. At this
point refinement was continued with the APS data set, and NCS
restraints were removed. Maximum Likelihood refinement with Refmac (39)
was interspersed with rounds of model building with XtalView (40).
Several applications of the wARP protocol (41) were used to identify
potential solvent molecules. A number of significant electron density
features were noted that had a tetrahedral shape and an appropriate
size to accommodate a sulfate or orthophosphate ion. Because the
crystal mother liquor contained 2.0 M LiSO4,
these peaks were modeled as sulfate ions. One of these sites, formed by
Arg27 and Arg60, approximates the binding of a
phosphoryl group of the DNA backbone as observed in the Co(II)-DtxR-DNA
complex (25). A total of 17 sulfate ions were included in the final model.
A)
versus resolution (37) for the final model were linear from
medium to the highest resolution. Val5, as discussed below,
is a severe outlier in the Ramachandran plot for all four IdeR
monomers. Arg60 is also a consistent but less severe
outlier in the Ramachandran plot. This might be functionally
significant because Arg60 is known to bind to a phosphoryl
group of the DNA backbone in the DtxR-DNA complexes. Residues
Ala148 and Asp149 of monomer A are also
outliers in the Ramachandran plot. This, however, seems to be due to
limitations of the data because these residues are in the linker region
between domain 2 and domain 3, which has very poor electron density.
Residues Asp147 and Asp148 of monomer A and
residue Arg194 of monomers C and D were truncated to their
C
atoms because of poor electron density. Final refinement
statistics for the APS data set are presented in Table I.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
angles fall within allowed regions of the
Ramachandran plot with 92.6% within the most favored regions (Fig.
1). Val5 falls into a
severely disallowed region of the Ramachandran plot (
= ~75°,
= ~
60°) in all four IdeR monomers. The
electron density revealed that this residue participates in a
turn
(48) from residues Leu4 to Asp6 (Fig.
2). The implications of this unusual
feature are discussed further below.
Data collection and refinement statistics
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Fig. 1.
Ramachandran plot for the final refined model
of the M. tuberculosis Co(II)-IdeR complex as produced
by PROCHECK. Valines 5 from all four monomers in the
asymmetric unit, labeled in red, are tightly clustered in a
disallowed region of -
space at about (+75°,
60°).
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Fig. 2.
Stereo diagram of the electron density of
the turn at Val5. The
A-weighted Fobs
Fcalc omit map was calculated after 20 cycles of
omit refinement with Refmac in Maximum Likelihood mode and is
contoured at 1.0
.
-strands and three
-helices. Superimposing 77 equivalent C
atoms from domain 3 of
IdeR monomer A and domain 3 of DtxR monomer 2 from the Co(II)-DtxR-DNA complex (25) resulted in an r.m.s. deviation of 1.38 Å. The linker
region (residues 141-150) between domain 2 and domain 3 is visible in
one IdeR monomer, although the electron density is generally poor, but
it is not discernible in the other three IdeR monomers.
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Fig. 3.
Stereo diagram of the unbiased electron
density for domain 3 of IdeR revealed by density modification and
4-fold improper NCS averaging. The C trace for the final
refined model is shown. The modified map was produced as described in
detail under "Experimental Procedures" and is contoured at 1.0
.
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Fig. 4.
Interface of domain 3 with domains 1 and
2. One monomer of IdeR is shown with its molecular surface
calculated by GRASP. The DNA binding helix, residues 38-52, is shown
as a ribbon. Co(II) atoms are shown as yellow
spheres and water molecules, labeled Sol, are
shown as blue spheres. Domain 3 is rotated out
and away from domains 1 and 2 for clear viewing, but the Co(II) atom
and tightly bound solvent molecule at metal site 1 are included in both
orientations for reference. Residues, excluding direct ligands to the
Co(II) atom at metal site 1, are shown whose centers make contacts
within 4.0 Å at the interface.
atoms for domains 1 and 2 (residues 1-140) were overlaid for each
possible pair of monomers, and the orientation of the domains 3 were
then compared. The difference in orientations is due to overall
rotations of 3.0-7.3° with the Co(II) atom at metal site 1 acting as
the approximate pivot point. Because all domains 3 are involved in
crystal contacts, it is difficult to assess the significance of this
observation. A similar analysis of domain 3 in the Co(II)-DtxR-DNA
complex (25), in which only one domain 3 is involved in a crystal
contact, showed differences in relative orientation ranging from 1.1°
to 8.0° (note that in this case three of the domains 3 are incomplete
so only residues 168-187 and 215-226 could be used in the
comparison). Taken together, these analyses indicate a certain degree
of flexibility in the interactions of domain 3 with domains 1 and 2.
A-weighted (37)
Fobs
Fcalc electron
density maps as a 13.3, 24.1, 17.7, and 9.9
peaks for the A, B, C,
and D monomers after the protein portion of the model was completely
built and partially refined. The site appears to be fully occupied with
refined B-factors for the Co(II) atoms of 21.3, 27.5, 32.1, and 30.5 Å2. The Co(II) atom is liganded by five protein
side chains; His79, Glu83, and
His98 are contributed by domain 2, and Glu172
and Gln175 are contributed by domain 3 (Fig.
5). The liganding atoms from each side
chain form direct coordinate bonds to the Co(II) atom (Table
II). Furthermore, there is no sixth
ligand; the Co(II) atom at metal site 1 is pentavalently coordinated
with distorted trigonal bipyramidal geometry. This geometry is a
common, fairly low energy state for Co(II), Fe(II), and other first row
transition metals (49) known to act as corepressors of DtxR (23).
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Fig. 5.
Metal site 1. Stereo diagram of the
A-weighted Fobs
Fcalc omit map calculated after 20 cycles of
omit refinement with Refmac in Maximum Likelihood mode and contoured at
1.0
. Residues of domain 2 are shown in orange, and
residues of domain 3 are shown in green. The Co(II) atom,
labeled 1, is shown as a yellow
sphere, and "water 1" bound in the anion site, labeled
Sol, is shown as a blue sphere.
Co(II)-ligand atom distances for metal binding sites
were identified near
metal sites 1 of the four IdeR monomers after refinement of the Co(II)
atoms. The peaks were identified as a water molecule by the wARP
protocol (41) and refined with B-factors of 19.9, 26.3, 26.8, and 33.3 Å2. This solvent-inaccessible Water 1 occupies an polar pocket formed by the side chains of
Arg80, Glu83, Ser126, and
Asn130 from domain 2 and Glu172, and
Gln175 from domain 3. Because of its pivotal position in
bridging the two metal binding ligands of domain 3 with domain 2, Water
1 may have an important role in metal-dependent activation
of IdeR.
peaks in
A-weighted Fobs
Fcalc electron
density maps. This site appears to be fully occupied with refined
B-factors for the Co(II) atoms of 23.2, 26.9, 31.1, and 31.4 Å2. The Co(II) atom is liganded by four protein side
chains: Met10 from domain 1 and Cys102,
Glu105, and His106 from domain 2 (Fig.
6). Cys102, which was
identified first by Tao and Murphy (22) as a crucial residue, forms a
bidentate interaction with the Co(II) atom; its S
and carbonyl
oxygen atoms form adjacent ligands in the octahedral coordination
sphere with an average angle of 88.0° between their coordinate bonds.
This is only slightly more acute than the ideal 90° angle for
octahedral coordination. The S
to metal ion distances range from
2.43 to 2.50 Å (Table II) and are within the range for metal-liganding
sulfur atoms (50).
View larger version (80K):
[in a new window]
Fig. 6.
Metal site 2. Stereo diagram of the
A-weighted Fobs
Fcalc omit map calculated after 20 cycles of
omit refinement with Refmac in Maximum Likelihood mode and contoured at
1.0
. Residues of domain 1 are shown in blue, and
residues of domain 2 are shown in orange. The Co(II) atom,
labeled 2, is shown as a yellow sphere, and water
2, which bridges the Co(II) atom to the main chain carbonyl of
Leu4, labeled Sol, is shown as a blue
sphere.
(17, 19). In this current structure of IdeR,
there is no sign of modification. In fact, this is the only refined
crystal structure of an iron-dependent regulator, aside
from the Co(II)-DtxR-DNA complex at 3.2 Å (25), in which the S
is
unambiguously making a direct coordinate bond to the metal atom.
peaks after refinement of the Co(II) atoms, was identified as a
water molecule by the wARP protocol, and refined to an average
B-factor of 29.4 Å2. Like the newly identified
Water 1 molecule at metal site 1, this Water 2 also appears to play a
role in the mechanism of metal-dependent repressor
activation. Water 2 bridges the cobalt atom at metal site 2 to the
N-terminal pentapeptide via the main chain carbonyl oxygen of
Leu4. The Co(II)-water distance is ~2.2 Å, and the Water
2-Leu4 carbonyl distance is ~2.8 Å.
turn (51) that reverses the main chain and
brings Val5 back to the dimer interface, where it packs
above Val107. Residues 4-6 form a hydrogen-bonded
turn
(48) that immediately reverses the polypeptide chain again. Although
the
turn forces the polypeptide main chain into the disallowed
region of
-
space according to early studies (52), later studies
show that this is in fact an allowed conformation (53).
View larger version (34K):
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Fig. 7.
Stereo diagram of the interaction of the
N-terminal residues with metal site 2. The Co(II) atom, labeled
2, is shown as a yellow sphere, and
the water molecule that bridges the Co(II) atom to the main chain
carbonyl of Leu4, labeled Sol, is shown as a
blue sphere. Side chains of residues discussed in
the text are colored blue and labeled. Electrostatic
interactions discussed in the text are shown as thick
red dotted lines. The hydrogen
bonds of helix 1 are shown for reference as thin
gray dotted lines.
-
conformation of
Val5 was noted in relation to the
Co(II)-water-Leu4 carbonyl interaction by White et
al. (24) for the Ni(II)-Cys102
AspDtxR-DNA and
Ni(II)-Cys102
AspDtxR structures and was also noted by
Pohl et al. (25) for the Co(II)-DtxR-DNA complex.
A-weighted Fobs
Fcalc electron density maps revealed an average
15.0-
peak adjacent to the N
2 atoms of residue His219
and His223 in domain 3 (which we will designate as metal
site 3) in all four IdeR monomers in the asymmetric unit. Because of
the peak intensity and its proximity to residues known for their
excellent metal liganding properties, these peaks were also modeled as
full occupancy Co(II) atoms that refined to an average
B-factor of 39.4 Å2. The separation of
His219 and His223 by a single turn of
-helix, much like Cys102 and His106 at metal
site 2, positions them to provide two adjacent ligands to the
octahedrally coordinated Co(II) atoms with an average 101.6° angle
between their coordinate bonds. The remaining four ligands are water
molecules that were identified by the wARP protocol, with the exception
of metal site 3 in the B monomer for which only three water ligands
were identified.
in the
A-weighted Fobs
Fcalc maps. The
interaction of each sulfate ion with IdeR is somewhat different.
2 atoms of
His212 from each of these three domains 3 converge upon an
oblong 11.6-
peak in
A-weighted
Fobs
Fcalc electron
density maps. This peak was modeled as a Co(II) atom, which refined to
a B-factor of 49.5 Å2, with two flanking water
molecules. A sixth ligand appears to be a partially occupied or
partially ordered water lying between the Co(II) atom and the main
chain carbonyl of Asp186 from monomer D. This interaction
highlights one notable difference between the monomers within the
asymmetric unit; the loop (residues ~207-217) containing
His212 in domain 3 appears in two distinct conformations.
In monomers A and D, this loop packs closely against domain 3 with
Ile207, Ile209, and Val215 making
hydrophobic contacts with Leu180, Leu184,
Ala187, and Val189. However, in monomers B and
C, this loop is rotated outward by ~25°. This Co(II) binding site
might be necessary for crystallization, but does not seem likely to be
important for repressor function.
atoms of
one domain 2 (residues 75-120) for each functional dimer of the
apo-DtxR (54) and the Co(II)-DtxR-DNA complex (25) onto one domain 2 of
the current Co(II)-IdeR structure (Fig.
8). The r.m.s. deviation in C
positions between the current Co(II)-IdeR structure and the
superimposed domains 2 is 0.40 Å for apo-DtxR and 0.39 Å for
Co(II)-DtxR-DNA. The r.m.s. deviation in C
positions for the domains
2 of the nonsuperimposed monomer is 0.70 Å for apo-DtxR and 0.56 Å for Co(II)-DtxR-DNA, indicating that the relative positions of domains 2 do not change significantly between these three structures. On the
other hand, the r.m.s. deviation in C
positions for the domains 1 of
the superimposed monomer is 1.63 Å for apo-DtxR and 1.34 Å for
Co(II)-DtxR-DNA, and for the nonsuperimposed monomer it is 2.06 Å for
apo-DtxR and 1.90 Å for Co(II)-DtxR-DNA. The DNA-binding domains among
these structures are thus moving considerably with respect to the core
of the dimer formed by the dimerization domains.
View larger version (47K):
[in a new window]
Fig. 8.
Superimposition of the apo-DtxR and
Co(II)-DtxR-DNA structures onto the Co(II)-IdeR structure.
Superimposition was performed as described in the text. The C trace
of Co(II)-IdeR is shown in blue, apo-DtxR in
green, and Co(II)-DtxR-DNA in orange. For
Co(II)-IdeR and Co(II)-DtxR-DNA the Co(II) atoms are also shown in the
corresponding colors. The hinge bending region near residue 74 is
indicated by black arrows, and the DNA-binding
helices and N termini are labeled for reference.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
of Cys102 has been confirmed to
be the fifth ligand to the Co(II) atom. The sixth ligand at metal site 2 was confirmed to be a tightly bound water molecule, Water 2, that
bridges the Co(II) atom to the main chain carbonyl of Leu4.
This Co(II)-water-Leu4 carbonyl interaction has been
previously observed by White et al. (24) in their 3.0-Å
resolution structure of the Cys102
Asp variant of DtxR
in complex with Ni(II) and DNA. White et al. proposed that
this bridging interaction was responsible, as a crucial aspect of
metal-dependent activation, for a helix-coil transition
that removed a severe steric clash between the N-terminal helix and
DNA, allowing DNA to dock properly to the DNA binding helices of domain
1. However, neither apo-DtxR solved in two crystal forms to ~2.3 Å by Pohl et al. (54) nor any of the previously determined
structures of DtxR with partially occupied or unoccupied metal sites 2 (17, 19) show a helical conformation of the N-terminal residues. The
poor electron density and typically high B-factors for the
N-terminal residues of these structures suggests that they are rather
flexible and disordered when metal site 2 is not occupied, and might
therefore be unable to maintain a rigid enough conformation to provide
a severe steric hindrance to DNA binding.
turn conformation of Val5 (Fig. 7). There is a helix
capping interaction between O
2 of Asp6 and the main
chain nitrogen atoms of residues Thr8 and Glu9
at the N terminus of helix 1. There are hydrogen bonds between O
1 of
Thr7 and N
2 of Asn2, and between the main
chain carbonyl of Asn2 and the main chain nitrogen of
Val5. The directional nature of these electrostatic
interactions might rigidify the conformation of the N-terminal
residues. Leu4 packs into a hydrophobic surface created by
the side chains of Met10, Tyr11, and
Leu34. Met1 is anchored at the dimer interface,
making hydrophobic contact with Val5 from both monomers of
the functional dimer. These hydrophobic interactions might enforce the
overall conformation and position of the N-terminal residues.
Interestingly, Leu4, Thr7, Tyr11,
and Leu34 are all highly conserved residues among the known
IdeR homologues (56). We propose that this represents the correct
conformation of the N-terminal residues for the active holorepressor
form of IdeR, and possibly for the active holorepressor form of DtxR as well.
AspDtxR-DNA
complex (24) and in the Co(II)-DtxR-DNA complex (25), the interaction
between domains 1 buried merely ~32 Å2 and ~12
Å2 of solvent-accessible surface area, respectively.
However, this comparison is impaired because in all of these previously
determined DtxR structures the N termini are incomplete, relatively
poorly defined in the electron density, and have in general very high B-factors. Thus, it is unclear what role the ordering of the
N-terminal residues might play in metal-dependent activation.
AspDtxR-DNA (24, 26) complexes.
In fact, the DNA binding helices of the functional dimers of
Co(II)-IdeR are shifted by 3.7-4.6 Å and rotated overall by
4.3-6.6° relative to the of DNA-binding helices of these two
DtxR-DNA complexes. The DNA-binding helices of Co(II)-IdeR are, stated
most simply, twisted somewhat into a more open conformation than those
in the DtxR-DNA complexes. The differences to the non-DNA-bound apo-
and metal-containing structures of DtxR solved previously are even
larger, ranging from a relative shift of 3.3 Å and relative rotation
of 7.1° for Zn(II)-DtxR (54) to a relative shift of 5.5 Å and
relative rotation of 13.4° for Ni(II)-Cys102
AspDtxR
(24). The orientation of the DNA-binding helices in the Co(II)-IdeR is
most similar to the described previously structure of Zn(II)-IdeR by
Pohl et al. (30), with a relative shift of 2.3 Å and
relative rotation of only 2.7°.
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ACKNOWLEDGEMENTS |
---|
We thank Stewart Turley for maintaining the in-house x-ray data collection equipment, Francis Athappilly for maintaining the Hol laboratory computing environment, and Joshua Gable for assistance with protein purification. We thank Keith Henderson at ALS and Rongguang Zhang at APS for assistance with synchrotron data collection.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01CA65656 (to W. G. J. H.) and R01AI14107 (to R. K. H.) and by a major equipment grant from the Murdock Charitable Trust (to the Biomolecular Structure Center at the University of Washington).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 1fx7) 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. E-mail:
hol@gouda.bmsc.washington.edu.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M007531200
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ABBREVIATIONS |
---|
The abbreviations used are: IdeR, iron-dependent regulator; DtxR, diphtheria toxin repressor; SH, Src homology domain; NCS, noncrystallographic symmetry; IRP, iron-regulated promoter/operator; APS, Advanced Photon Source; ALS, Advanced Light Source; r.m.s., root mean square; CC, correlation coefficient; MES, 4-morpholineethanesulfonic acid.
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