From the Department of Biological Sciences,
University of Calgary, Calgary, Alberta T2N 1N4, Canada,
§ Syrrx Inc., San Diego, California 92121, and
the ¶ Department of Microbiology and Infectious Diseases,
University of Calgary, Calgary, Alberta T2N 4N1, Canada
Received for publication, November 19, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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The periplasmic iron binding protein of
pathogenic Gram-negative bacteria performs an essential role in iron
acquisition from transferrin and other iron sources. Structural
analysis of this protein from Haemophilus influenzae
identified four amino acids that ligand the bound iron:
His9, Glu57, Tyr195, and
Tyr196. A phosphate provides an additional ligand, and the
presence of a water molecule is required to complete the octahedral
geometry for stable iron binding. We report the 1.14-Å resolution
crystal structure of the iron-loaded form of the H. influenzae periplasmic ferric ion binding protein (FbpA) mutant
H9Q. This protein was produced in the periplasm of Escherichia
coli and, after purification and conversion to the apo form, was
iron-loaded. H9Q is able to bind ferric iron in an open conformation. A
surprising finding in the present high resolution structure is the
presence of EDTA located at the previously determined anion ternary
binding site, where phosphate is located in the wild type holo and apo
structures. EDTA contributes four of the six coordinating ligands for
iron, with two Tyr residues, 195 and 196, completing the coordination. This is the first example of a metal binding protein with a bound metal·EDTA complex. The results suggest that FbpA may have the ability to bind and transport iron bound to biological chelators, in
addition to bare ferric iron.
Iron plays a key role in many essential biological
processes (1). Therefore, the acquisition of iron is integral for the survival of almost all organisms. Due to the low solubility of free
ferric iron (Fe3+) under physiological conditions, a
variety of specialized systems for uptake, transport, and storage of
this metal ion cofactor have been developed by living organisms (2).
The majority of the iron obtained through the dietary intake in humans
is stored and utilized in the intracellular compartment (3).
Extracellular iron is complexed by the bi-lobed transport glycoprotein,
transferrin (Tf),1 within the
body or by lactoferrin (Lf) on mucosal surfaces (4). These iron binding
glycoproteins are produced in excess of free iron and bind iron at
extremely high affinities (Kd = 1 × 10 The ability of a pathogen to colonize and grow within the host is
essential for the initiation of an infection (7). Thus to cause disease
the pathogen must be successful in competing for protein bound iron in
the host. In bacterial pathogens the expression of genes encoding iron
acquisition proteins results from an adaptation to the iron-limiting
environment within the host (8). Bacteria have diverse and elaborate
systems capable of scavenging iron from host proteins. Some pathogenic
microorganisms are able to synthesize and secrete low molecular
mass (500-1000 Da) organic iron chelators termed siderophores,
which are able to bind iron with association constants as high as
1053 (3, 9). This mechanism of iron acquisition is
effective for a variety of different iron sources and environments and
is typically found in bacteria that are present in a variety of
different ecological niches. Iron is transported into the bacterial
cell in the form of an iron chelate by this mechanism (9).
Another strategy is chelate-independent and employs an array of
distinct outer membrane receptors, which specifically interact with the
different host iron binding proteins. This mechanism is utilized by
pathogenic bacteria of the Neisseriaceae (e.g. Neisseria gonorrhoeae and Neisseria meningitidis)
and Pasteurellaceae (e.g. Haemophilus influenzae)
families, which are not capable of producing siderophores (10, 11).
These microorganisms produce Tf and Lf surface receptors that are
capable of binding the iron-loaded host proteins as the first step in
this elaborate iron acquisition system (12). The heterodimeric receptor
complex typified by the Tf receptor is composed of two proteins,
transferrin binding proteins A and B (TbpA and TbpB, respectively).
Even though the binding of iron-containing proteins to the bacterial
surface receptors is well characterized, the subsequent steps of iron
removal and transport into the bacterial cell are not well understood.
The removal of iron from Tf and the subsequent transport of free iron
across the outer membrane requires energy provided by TonB and its
associated proteins, ExbB and ExbD (13). Once the iron is removed from
Tf, and transported across the outer membrane, it is bound by the
periplasmic ferric ion binding protein (FbpA) (14). The continued
transport of Fe3+ from FbpA across the cytoplasmic
membrane requires the genetically linked hydrophobic
membrane protein, FbpB and the cytoplasmic membrane-associated
nucleotide binding protein, FbpC, which together form a
Fe3+ ATP-binding cassette transporter (15). Once inside the
cytoplasm ferric iron is able to bind iron-coordinating proteins
required for growth or be stored in the form of bacterioferritin
(16).
A series of site-directed mutants of FbpA were prepared to evaluate the
role of the various amino acid side chains in metal binding and their
importance for the iron acquisition process. In this study, we report
the 1.1-Å nominal resolution and 1.14-Å actual resolution (where the
last shell is >50% complete) crystal structure of a holo H. influenzae site-directed mutant (H9Q) FbpA in complex with EDTA.
With EDTA bound at the previously described anion-binding site, FbpA
adopts an open conformation. Characteristics of the residues involved
in EDTA binding show how FbpA may be able to accommodate a broad range
of anion ligands. The EDTA molecule is able to fulfill the octahedral
requirements of Fe3+ by contributing four of the required
six ligands. This is the first example of a metal binding protein,
which binds a metal·EDTA complex.
Purification and Crystallization
Procedures--
Escherichia coli BL21(DE3)/pLysS harboring
the pT7-7 plasmid encoding FbpA with the His9 to Gln
mutation (H9Q) was used to express the H. influenzae mutant protein in the periplasm under
isopropyl-1-thio-
Following dialysis, the protein sample was subjected to cation exchange
chromatography on the Bio-Cad high-performance liquid chromatography system as a final purification step. The column was
washed with 10 volumes of 20 mM Tris/HCl buffer, pH 7.5, to remove unbound proteins and eluted with a gradient of 0-1.5
M NaCl. The material collected from the column was
concentrated to a final volume of between 1-3 ml using a Centricon 10 microconcentrator. The removal of iron from the protein was then
accomplished by treatment of the protein in the Centricon 10 microconcentrator with 1 mM EDTA and a 4000-fold molar
excess of citrate (sodium salt, pH 7.5) and incubation on ice for 20 min. At this point the protein, still in the Centricon, was
exhaustively exchanged into 10 mM Tris/HCl buffer, pH 8.0. Once completed, 5 mM phosphate was added to the Centricon,
and the sample was again incubated on ice for 20 min. After the
incubation period, it was again washed extensively with 10 mM Tris/HCl buffer, pH 8.0. Finally, the sample was
concentrated to ~25 mg/ml and iron was loaded. Iron-saturated FbpA was prepared by adding a 5-fold molar excess of a ferric citrate
solution. A fresh ferric citrate solution was prepared by dissolving
ferric chloride (FeCl3·6H2O, Sigma) in 100 mM sodium citrate/100 mM sodium bicarbonate
buffer. The protein and ferric citrate solution was then placed at
4 °C overnight before being used for crystallization or being frozen
for later use. The resulting preparations were deemed pure based on
SDS-PAGE analysis. All proteins were stored at
The sitting-drop vapor diffusion method was employed for
crystallization of the protein. All crystallization experiments were conducted at 20 °C with 4-µl drops containing Fe-FbpA, 10% (w/v) PEG 5000, and 50 mM Tris/HCl buffer solution at pH 8.2. The
drops were equilibrated against a 1-ml reservoir containing
20% PEG 5000 and 100 mM Tris/HCl buffer, pH 8.2. Large,
pink crystals of the protein grew within 1 week. These crystals were
suitable for x-ray diffraction and belonged to the orthorhombic space
group P21212 (a = 105.1 Å,
b = 75.3 Å, c = 33.6 Å).
Data Measurement--
The crystals in a sitting drop were
harvested by scooping them with a nylon loop. The crystal was then
dipped into a cryoprotectant solution containing 30% (v/v) ethylene
glycol, 100 mM Tris/HCl buffer, pH 8.2, and 20% (w/v) PEG
5000 for ~30 s before they were frozen in liquid nitrogen.
Diffraction data were collected from a frozen crystal of the H9Q
FbpA·EDTA·Fe complex up to 1.1 Å at 100 K using a QUANTUM4
charge-coupled device camera on Beamline 5.03 at the Advanced Light
Source Berkeley Laboratory, Berkeley, CA, using a wavelength of
1.0 Å. The data were reduced and scaled using the HKL2000 software
(17). The dataset statistics are given in Table I.
Structure Determination--
The crystal structure was
determined by a molecular replacement procedure using the program
Molrep from the CCP4 suite of programs (18). The coordinates of the
H. influenzae wild type apoFbpA (open conformation) were
used as the search model. Iterative cycles of interactive manual
refitting of the model using the program XtalView/xfit made use of maps
created with ARP/wARP 5.2 and refinement with Refmac5 was carried out
to complete and correct the model. During the later stages of
refinement, difference maps (Fo Structure Analysis--
The overall structure obtained for the
mutant FbpA is similar to the previously reported metal-loaded and
apoFbpA structures. The refined coordinates of H. influenzae
H9Q FbpA in complex with EDTA·Fe have been deposited in the Protein
Data Bank. Ramachandran plots of the mutant structure shows the
satisfactory location of all residues into allowed regions of
conformational space. The DALI server (www2.ebi.ac.uk/dali) was
utilized to find structurally similar proteins in the Fold
Classification Based on Structure-Structure Alignment of
Proteins data base (19).
Crystal Production--
The recombinant H9Q FbpA mutant protein
was isolated from the periplasm of E. coli and purified by
ion-exchange chromatography. Following conversion to the apo form, the
protein was exhaustively exchanged into a 10 mM Tris/HCl
buffer, pH 8.0. The production of an apo form allowed for the
possibility of various metals to be added to the sample to determine
binding affinities for the mutant protein (results not shown). A
portion of the concentrated apo sample was iron-loaded with a 5-fold
molar excess of ferric ions dissolved in a citrate/bicarbonate buffer
for crystallization. Crystals grew within 1 week using the sitting drop
method with 4-µl drops and a 1-ml reservoir of 20% PEG 5000 and 100 mM Tris/HCl buffer, pH 8.2, at 20 °C.
Structural Features--
H9Q FbpA has the same overall topology as
the apo form of wild type FbpA, with approximate dimensions of 60 Å × 30 Å × 40 Å (Fig. 1). The mutant
protein, like the wild type FbpA is composed of two structural domains,
termed the N and C domains. Each domain is composed of a twisted
five-stranded mixed
Comparison of the H9Q FbpA crystal structure with that of the H. influenzae wild type FbpA structures reveals that the mutant protein is able to bind iron in an open conformation (Fig. 1). The C
The crystallographic refinement parameters (Table
I) and the final electron density maps
and Ramachandran plot (20) (not shown) of the main chain torsion angles
as defined by the program PROCHECK (21) indicate that the crystal
structure of H9Q is of high quality. In the Ramachandran plot 94.2% of
the non-glycine residues are in the most favored regions and 5.8% are
in additional allowed regions. This structure represents the first
example of a metal binding protein with a bound metal·EDTA
complex.
Iron and EDTA Binding Sites--
Electron density maps showing the
bound EDTA molecule and iron atom are displayed in Fig.
3. The high resolution electron density
maps (2 Fo
A diagram of the EDTA·Fe binding site is displayed in Fig.
4. In this H9Q FbpA structure, the iron
binding residues of the C-terminal domain (Tyr195 and
Tyr196) adopt essentially identical placements and
conformations with respect to the iron atom as those in the wild type
holoFbpA structure (22). However, the iron binding residues of the
N-terminal domain (Gln9 and Glu57) adopt
similar conformations to the wild type apoFbpA (23). These residues
have rotated away from their iron binding locations and are
hydrogen-bonded to residues within their N-terminal domain across the
groove from the iron atom. The distances of Glu57-O
Filling the role of these two residues in the coordination of iron is a
bound EDTA molecule. Because the EDTA is a rather large molecule,
containing two nitrogen atoms and four carboxyl groups, it is also able
to fulfill the roles of the phosphate anion and the water molecule that
are found in the holoFbpA structure (22). The EDTA wraps around the
iron atom, and one of the carboxyl groups is able to bind in the
previously described ternary anion-binding site. As illustrated in Fig.
4, the EDTA provides four of the six coordinating ligands to iron
binding. Thus, four oxygen donors and two nitrogen ligands still
octahedrally coordinate the bound Fe3+. The ligands include
Tyr195-OH, Tyr196-OH, two EDTA oxygen atoms
(O14 and O19), and the last two sites may be weakly coordinated by EDTA
nitrogen atoms (N3 and N8) or almost empty, because the distances of
these ligands from the iron are slightly longer (2.88 and 2.52 Å,
respectively) when compared with the other coordination distances and
distances previously reported for EDTA nitrogen atoms to
Fe3+. The iron coordination is distorted from perfect
octahedral geometry, with distances ranging from 1.92 to 2.88 Å from
iron to the coordinating ligands (Table
II).
Previous small molecule crystallography on an EDTA·Fe complex
revealed similar ligation geometry to that observed in the H9Q structure (24). The oxygen atoms in the small molecule structure that
represent Tyr195-OH and Tyr196-OH in the H9Q
structure have ligation distances of 1.98 and 2.10 Å, respectively.
Also the oxygen atoms of EDTA in the small molecule structure that
correspond to the coordinating EDTA oxygens (O14 and O19) in the H9Q
FbpA have ligation distances of 2.10 and 1.97 Å, respectively.
Although the above distances for oxygen atoms are very similar to those
reported in Table II, the distances to the liganding nitrogen atoms in
the H9Q structure are slightly longer than the previously reported
distances of 2.32 and 2.32 Å for the same residues in the small
molecule complex. The bond angles in the small molecule complex range
from 71.8° to 106.0° (24). In H9Q the angles involving the nitrogen
atoms deviate slightly from this range adding to the speculation that
these residues may be coordinating the Fe3+ very weakly.
The EDTA molecule is well ordered with an average
temperature factor of 17 Å2. The binding of EDTA is
stabilized by the location of one of the carboxyl groups in the
previously determined anion-binding site. This carboxyl group is held
in place by a similar constellation of residues as are shown to bind
phosphate in the wild type apo and holo forms. The residues that
contribute hydrogen bonds to this carboxyl group of EDTA include
Asn175, Asn193, Ser139, and
Tyr195. With a putative hydrogen bond also forming between
the backbone nitrogen atom of Ala141 and the carboxyl group
of EDTA. The other carboxyl group involved in the coordination of the
iron interacts with the protein though hydrogen bonds with the side
chain of Arg262. These data suggest that there is a high
degree of promiscuity in anion binding by FbpA and that FbpA is likely
able to bind iron complexed with a wide range of chelators.
Analysis of the H9Q FbpA structure reveals that it adopts the
typical periplasmic ligand binding protein (PLBP) fold that has been
observed for almost all structurally determined PLBPs to date. Although
PLBPs recognize a large variety of ligands and have very little
sequence homology, almost all PLBP structures known to date are
composed of two globular domains with the binding site located in a
groove between these domains. The domains are connected by a number of
flexible Due to the striking structural similarity between transferrin and many
periplasmic binding proteins, these proteins have been included in the
transferrin structural superfamily (22). This group of proteins
includes the eukaryotic transferrins, lactoferrins, and the bacterial
periplasmic binding proteins, which transport a diverse group of
ligands in the periplasmic space of bacteria. Despite operating in
different organisms and cellular locations, human Tf and the H. influenzae FbpA appear to be structurally and functionally
homologous (22). FbpA shares many structural similarities with each
lobe of Tf, including two globular domains, each centered on a twisted
mixed Previous x-ray crystallographic analyses have illustrated that for
those proteins of the transferrin structural superfamily for which both
the apo and holo forms have been solved, the two domains rotate toward
each other in the liganded structure (closed conformation) (8). The apo
form of FbpA has the same overall topology as the holo form, however,
the two domains of the protein are rotated about 20° with respect to
each other (Fig. 1). A change in the dihedral angles of the
antiparallel Our initial intent was to determine the role of the metal-liganding
residue His9 in the coordination of iron in the H. influenzae FbpA. Site-directed mutagenesis was used to convert the
histidine residue to a glutamine amino acid. In a companion study, the
resulting mutant protein was tested for its metal-binding properties
and ability to support transport of iron from
transferrin.2 As revealed by
the present crystal structure the H9Q mutation has not affected the
protein folding of FbpA (Fig. 1). However, the most interesting
information gleaned from the H9Q structure is the protein's ability to
bind iron in an open conformation with the use of an EDTA molecule. The
presence of a bound EDTA molecule was very surprising given that EDTA
was added only to convert the FbpA into an apo form and was not present
in any subsequent solutions.
This high resolution structure provides valuable information for the
structural pathway illustrating the conformational transition from the
open to closed forms upon iron binding. The FbpA is fully iron-loaded,
with the ordered iron-binding site of the C-domain occupied; yet the
protein remains in an open conformation most likely due to the location
of the EDTA molecule in the binding cleft. A similar structural state
was reported for transferrin when crystallized with ferric
nitrilotriacetic acid (29). Due to the fact that this
FbpA·iron·EDTA complex formed in solution prior to crystallization,
because EDTA and iron were absent from the crystallization conditions,
two models can be proposed.
In the first model, EDTA binds in a mode that was first proposed for
phosphate, based on the apo and holo structural information (22, 23).
The EDTA molecule would likely be sequestered in the anion-binding site
and close over the top of the iron once Fe3+ bound to the
tyrosines. The other carboxylate group that is ultimately involved in
iron coordination would likely have to remain free in this model until
the Fe3+ is bound in order for the iron to have access to
the tyrosines. This model is supported by the fact that during sample
preparation the protein was washed extensively with buffer after which
a large excess of phosphate was added to the sample, which was
subsequently washed repeatedly with buffer again. The lack of phosphate
in the present structure suggests that EDTA may have occupied the anion-binding site when phosphate was added to the solution and the
favorable hydrogen bonds maintained the EDTA in position even during
the buffer exchange protocol.
The second model assumes that the iron binds to the protein followed by
the EDTA anion. In this model EDTA, either free in solution or
associated with the protein at some other location, initially binds
free ferric iron and then moves to the highly ordered iron-binding site
involving the tyrosine residues (Tyr195 and
Tyr196) of the C domain or wraps over the top of the
Fe3+ after the iron has bound to the tyrosine residues. Due
to the steps involved during the sample preparation, the metal·EDTA
complex would likely have to displace phosphate at the anion-binding
site in this scenario. Regardless of the order involved, the present high resolution structure illustrates the point that ferric iron initially associates with the ordered tyrosine residues
(Tyr195 and Tyr196) of the C domain. The two
domains would then rotate together as the histidine and glutamic acid
residues are able to complete the coordination of free ferric iron in
the wild type protein (22). The H9Q structure reveals how FbpA may be
able to accommodate other metal· anion complexes.
A very important observation obtained from the H9Q structure with EDTA
is the ability of a carboxyl group to occupy the anion-binding site.
This was surprising, because the protein was also exposed to phosphate
prior to the iron loading process. Previously it had been shown that a
phosphate molecule was located at this ternary binding site (22, 23).
The carboxyl group of EDTA found at the anion binding site forms
hydrogen bonds with many of the same amino acids the phosphate anion
was shown to bond to (Fig. 4). In the wild type holo form,
PO The observation of a carboxylate group occupying the anion-binding site
rather than the previously determined phosphate is consistent with
functional studies that demonstrated that a number of oxyanions can
fill the role of the ternary anion (23, 30). Other groups have also
discussed the ability of H. influenzae to grow in the
presence of a number of iron chelates, with the present study providing
an explanation as to how transport of these iron complexes may occur
(31). This structure reveals how FbpA is able to not only bind free
ferric iron but how it may be able to accept a variety of anions under
physiological conditions.
It appears that the mutation of His9 to Gln has very little
effect on the present structure. The Gln residue adopts a very similar
conformation to that of His in the apo wild type structure. The Gln
residue rotates away from the iron binding site and hydrogen bonds with
other residues present in the N-terminal domain. The glutamine is
located across the cleft 6.05 Å away from the iron atom. Obviously,
the Gln residue is unable to bind iron in the presence of EDTA due to
steric hindrance. However, there seems to be no structural reason a Gln
side chain could not coordinate to iron, as in theory it could
coordinate iron through its amide oxygen. Primary sequence alignment of
FbpA sequences from H. influenzae and Pasteurella
hemolytica reveal that the histidine residue is replaced by a
glutamine in the P. hemolytica protein. In fact, the crystal
structure of the same histidine to glutamine mutation has been
determined for the N-lobe of transferrin showing that this mutation is
still able to octahedrally coordinate iron (32). However, it must be
noted that the H249Q Tf structure may exist in the closed state, due to
the increased number of interdomain interactions present in Tf that do
not exist in the FbpA, because of to its more solvent-exposed cleft.
Even though the H249Q transferrin protein is still able to coordinate
iron in a closed conformation, spectroscopic studies have described
altered properties, both in the acid stability of iron binding and the
kinetics of iron release to chelators indicating a reduced affinity for
iron (33). Thus, no conclusions can be reached at this point as to
whether the H9Q FbpA would be a functional protein in the bacterium.
In conclusion, we have determined the first high resolution crystal
structure of a metal binding protein, which binds a metal·EDTA complex. The previous crystallographic structures of FbpAs have been
restricted essentially to the two structural forms: the first is the
apo or open form, and the other is the holo or closed form (22, 23). As
an alternate structural state, the current EDTA·Fe3+
structure is the first demonstration of an iron-loaded, open FbpA form.
The high resolution structure allows for three important observations.
First, only the two tyrosine residues (Tyr195 and
Tyr196) participate as protein ligands in the coordination
of the iron atom. This observation provides evidence as to the entry of
iron into the apo protein. Second, the presence of the carboxyl group stably bound in the anion-binding site instead of a phosphate anion,
supports the idea of FbpA being able to use other available anions from
the periplasmic space to coordinate iron. Finally, there is the
observation of the ability of FbpA to bind a large molecule (EDTA) in
the relatively shallow binding groove. The final observation proposes
many more questions than answers. At this point in time we are unable
to comment fully as to the significance of such a finding. Does this
mean that wild type FbpA is able to accept and transport other iron
complexes rather than just free ferric iron? Obviously, future studies
on the H9Q and wild type proteins will have to explore this
possibility. With such a shallow binding pocket compared with the
transferrins, it is not unreasonable to speculate that FbpA may be able
to bind biological iron chelate complexes, such as ferric citrate for
transport into the bacterial cell. From the present study it is
apparent that FbpA can accommodate much larger anions than phosphate.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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21 M), effectively reducing the
availability of this element, and thus protecting against bacterial
infection (5, 6).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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-D-galactopyranoside induction. The
protein was then purified using a modified osmotic shock procedure.
Briefly, the broth culture was harvested by centrifugation at 4000 rpm
for 20 min using a GSA rotor at 4 °C. The supernatant was
discarded, and each pellet was resuspended in 1/10 volume (50 ml) of
buffer composed of: 30 mM Tris/HCl buffer, pH 8.0, 20%
sucrose, and 1 mM EDTA. The solution was rocked for 10 min at room temperature. The samples were then centrifuged at 4000 rpm for
30 min using a GSA rotor at 4 °C. The supernatant was again
discarded, and the bottles were inverted onto paper towels briefly to
remove all traces of supernatant. The pellets were rapidly resuspended
in 10 ml of ice-cold 5 mM MgSO4 by pipetting up
and down with a 1-ml pipette. The solutions were left on ice for 20 min. After which time the samples were centrifuged at 4000 rpm for 30 min using a GSA rotor at 4 °C. The supernatant was carefully
removed, without disrupting the pellet, because the supernatant
contains the periplasmic osmotic shock fraction. Finally, the sample
was extensively dialyzed against 10 mM Tris/HCl buffer, pH
8.0, at 4 °C.
80 °C until they
were used for crystallography.
Fc maps) were used to place the bound EDTA·iron complex. Restrained refinement using a maximum likelihood target function and anisotropic temperature factors for individual atoms was carried out. During the final refinement stage, well-defined residual electron density peaks in difference maps were assigned to
water molecules. The last residue of the C terminus was not visible in
the electron density map, so it was omitted. Several side chains of
surface-exposed residues are also missing (residues 10, 11, 27, 43, 93, 119, 244, and 282) and were built as alanines. The final
crystallographic R-factor is 0.158 for all data from 105.4- to 1.1-Å resolution. The free R-factor for randomly
selected 5% data is 0.180.
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-sheet surrounded by
-helices (Fig.
2). The two domains are connected by two
antiparallel
-strands, which form the hinge between the two domains.
Hinge bending between the two domains enables the participation of both domains in the binding and sequestering of the EDTA·Fe ligand.
View larger version (40K):
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Fig. 1.
Stereo C plots of
H. influenzae FbpA. The apo (yellow)
and holo (blue) forms of the wild type protein are
superimposed with the H9Q structure (green). The figure was
produced with XtalView/Xfit (34) and ViewerLite 5.0 (available at
www.accelrys.com). The models are superimposed on the C domain. The
holo and apo structures were derived from their respective pdb files
(1MRP and 1D9V) from the Protein Data Bank. 1D9V was employed as the
model for the current structural determination of the H9Q form. The
iron-binding tyrosine residues (Tyr195 and
Tyr196) of the C domain are shown in pink, and
the iron atom is in orange. The N-terminal domain is at the
bottom.
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Fig. 2.
Overall structure of H9Q FbpA complexed with
EDTA·Fe. A stereo ribbon diagram showing the overall
tertiary structure and secondary structure elements in the complex. The
EDTA molecule and iron atom are black. Helices, -strands,
and random coil regions are gray. This figure was produced
using XtalView/Xfit (34) and ViewerLite 5.0. The N and C termini are
labeled.
trace shows that the mutant protein form is highly similar to the apo
wild type form. The root mean square deviation for 308 C
atoms is
only 0.50 Å, whereas the root mean square deviation for the same 308 C
atoms of the apo and holo structures is 5.55 and 5.76 Å when the
mutant and wild type holo forms are compared.
Summary of crystallographic data collection and refinement
Fc
c and Fo
Fc
c) resolving individual atoms
allowed for the unambiguous placement of the EDTA molecule at the
binding site. Other anions were considered based on buffers used during the purification and crystallization protocols, but they were later
rejected due to their lack of agreement with the electron density
maps.
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Fig. 3.
Stereo view showing the iron-binding site of
the H9Q FbpA. Electron density maps (green,
2 Fo Fc
c
map, contoured at 1
; purple, Fo
Fc
c map, contoured at 2
) were
obtained using reflection data in which the EDTA and iron residues were
omitted. This figure was made using XtalView/Xfit (34) and Raster3D
(35).
1 and
Gln9-O
1 from iron are 9.19 and 6.05 Å,
respectively.
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Fig. 4.
Diagram of the Fe3+ coordination
and hydrogen bonding network. a, the EDTA form;
b, the holo form. The figure for the holo form is drawn
using previous data (22) for comparison. The thick solid
lines in black represent the possible coordination to
Fe3+, although the coordination by EDTA-N (3) and -N (8)
are not clear because of longer iron to ligand distances than the other
coordination distances (see Table II). The dashed lines in
black display hydrogen bonds with the bond distances in
angstroms. The synergistic anions (EDTA in a and phosphate
in b) are shown in gray.
Geometry of the iron binding site
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-strands at the bottom of the ligand-binding site (25, 26).
Even though FbpA possesses the typical PLBP structure, none of the
other closely related periplasmic transport proteins bind naked metal
atoms (27). A search using the DALI (19) server for proteins in the
Protein Data Bank with similar three-dimensional folds to FbpA reveals
structural similarity with sulfate binding protein, maltodextrin
binding protein, spermidine binding protein, phosphate binding protein,
and the N-terminal half-molecule of ovotransferrin.
-sheet surrounded by
-helices. In each protein a
"hinge," which is composed of antiparallel
-strands, connects
the two domains. Moreover, the coordination of the Fe3+ by
the oxygens of two tyrosines, an imidazole nitrogen from histidine and
a carboxylate oxygen from an acidic residue (Asp in Tf and Glu in
FbpA), is identical (28). However, there are also some important
structural differences between Tfs and FbpA. The first involves how
each protein is able to complete the octahedral coordination of
Fe3+. Transferrin utilizes two oxygens from a carbonate
anion, whereas FbpA makes use of an oxygen from a phosphate and another
from a water molecule (22). Although the subset of amino acids
coordinating the iron atom is very similar, the residues arise from
different regions of the individual proteins. The iron-binding site is
also more solvent-exposed in FbpA than it is in transferrin. The
discrepancy in iron exposure is due to the binding site being shallower
in FbpA and the absence of an additional loop covering the binding site
in Tf.
-strands in the joining hinge region opens the binding
site to the solvent.
2, Asn175-N
2,
Ser139-O
, Ala141-N, and
Gln58-O
1. With the exception of Gln58-O
1,
all of these moieties are hydrogen-bonded to the carboxylate group that
occupies the anion-binding site. The other carboxylate group that is
involved in the iron coordination is stabilized by hydrogen bonds to
the protein residue Arg262. The remaining two carboxylate
groups of the EDTA molecule point out of the binding cleft and are
hydrogen-bonded to water molecules. These groups of the EDTA molecule
do not interact with protein residues in any way.
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ACKNOWLEDGEMENT |
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We are indebted to Syrrx, Inc. for allowing the use of laboratory space and beam time toward the completion of this project.
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FOOTNOTES |
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* This work was supported by Grant 49603 from the Canadian Institutes for Health Research.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 1NNF) 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
Microbiology and Infectious Diseases, University of Calgary, Rm. 274, Heritage Medical Research Bldg., 3330 Hospital Dr. NW, Calgary, Alberta
T2N 1N4, Canada. Tel.: 403-220-3703; Fax: 403-270-2772; E-mail:
schryver@ucalgary.ca.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M211780200
2 A. G. Khan, S. R. Shouldice, R. Yu, and A. B. Schryvers, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Tf, transferrin; Lf, lactoferrin; FbpA and -B, ferric ion binding proteins A and B; TbpA and -B, transferrin binding proteins A and B; PLBP, periplasmic ligand binding protein; PEG, polyethylene glycol.
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