From the School of Chemistry, University of
Edinburgh, Edinburgh EH9 3JJ, United Kingdom, ¶ Synchrotron
Radiation Department, CLRC Daresbury Laboratory, Warrington WA4 4AD,
United Kingdom,
Department of Biochemistry and Molecular
Biology, University of British Columbia, Vancouver, British
Columbia V6T 1Z3, Canada, and ** Department of Chemistry,
University of Missouri, St. Louis, Missouri 63121
Received for publication, August 27, 2002
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ABSTRACT |
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The 34-kDa periplasmic iron-transport protein
(FBP) from Neisseria gonorrhoeae (nFBP) contains Fe(III)
and (hydrogen)phosphate (synergistic anion). It has a characteristic
ligand-to-metal charge-transfer absorption band at 481 nm. Phosphate
can be displaced by (bi)carbonate to give Fe·CO3·nFBP
( Iron is an essential nutrient absolutely required for all
organisms with the possible exception of Lactobacillus and
Borrelia burgdorferi (1, 2). Iron is abundant in the
environment but tends to precipitate as ferric hydroxide under aerobic
aqueous conditions at neutral pH where the concentration of free
Fe(III) is 10 FBP has been called "bacterial transferrin" because of its
remarkable similarity to transferrin. A recent crystal structure of FBP
from H. influenzae (hFBP) has revealed that it is
structurally homologous to one lobe of transferrin but without sequence
homology (9). The Fe(III) binding site in hFBP resembles the two
Fe(III) binding sites in transferrin containing two cis-Tyr
ligands and one His ligand but with Glu instead of the Asp residue
found in transferrins (see Structure
I). A striking difference is that hFBP appears to have a monodentate tetrahedral phosphate (or hydrogen phosphate) bound to Fe(III) as the synergistic anion together with a
water ligand (9), whereas transferrins use bidentate (bi)carbonate as
the synergistic anion. Transferrin can use other carboxylates as
synergistic anions but cannot use phosphate or sulfate (13). The
synergistic anion binding properties of transferrins including their
role in metal binding and release have been well documented (14-16),
but only recently have the synergistic anion binding properties of
bacterial FBP been investigated (9, 17). The FBP from N. gonorrhoeae (nFBP) has been biochemically characterized (18) and
subsequently expressed in Escherichia coli (19, 20). It has
been reported that a large excess of NaHCO3 increases the iron binding capacity of apo-FBP (21).
max 459 nm). The local structures of native Fe-PO4-nFBP and Fe·CO3·nFBP were determined
by EXAFS at the FeK edge using full multiple scattering analysis. The
EXAFS analysis reveals that both phosphate and carbonate ligands bind
to FBP in monodentate mode in contrast to transferrins, which bind
carbonate in bidentate mode. The EXAFS analysis also suggests an
alternative to the crystallographically determined position of the Glu
ligand, and this in turn suggests that an H-bonding network may help to stabilize monodentate binding of the synergistic anion. The anions oxalate, pyrophosphate, and nitrilotriacetate also appear to serve as
synergistic anions but not sulfate or perchlorate. The oxidation of
Fe(II) in the presence of nFBP led to a weak Fe(III)·nFBP
complex (
max 471 nm). Iron and phosphate can be removed
from FBP at low pH (pH 4.5) in the presence of a large excess of
citrate. Apo-FBP is less soluble and less stable than Fe·nFBP and
binds relatively weakly to Ga(III) and Bi(III) but not to Co(III) ions,
all of which bind strongly to apo-human serum transferrin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
18 M, 12 orders of magnitude
lower than that required for bacterial growth (3, 4). In humans and
other mammals, iron is sequestered and transported by transferrins and
lactoferrins and stored in ferritin (5). The tight binding of iron by
these proteins leads to a very low level of free iron in the host,
which acts as an antibacterial defense mechanism. However, some
pathogenic bacteria, such as Neisseria meningitidis,
N. gonorrhoeae, Haemophilus influenzae, Actinobacillus actinomycetemcomitans, and Pasteurella
hemolytica, have developed highly efficient iron-uptake systems
including specific iron-binding proteins
(FBPs)1 to sequester iron
from hosts (6, 7). Iron is transported in the periplasm of these
pathogens by FBP, which is a highly conserved periplasmic protein
required for virulence (7-12). Therefore, structural studies of FBP
are important for understanding the iron-uptake mechanism in these
pathogens and could aid the design of novel bacteriostatic agents
targeted on this protein.
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Structure 1.
Fe(III) binding site in FBP. The
state of protonation of phosphate is unknown.
We have investigated the binding of anions and metal ions to
recombinant FBP from N. gonorrhoeae by a variety of
techniques including UV-visible spectroscopy, inductively
coupled plasma atomic emission spectrometry (ICP-AES), electrospray
mass spectrometry, multinuclear NMR spectroscopy, and EXAFS.
EXAFS is a technique capable of providing, in principle, metal-ligand
bond lengths to the same accuracy as a small molecule crystal structure
(0.02 Å). In the case of metalloproteins, this accuracy
significantly exceeds the accuracy obtained from all but atomic
resolution (<1.2 Å) crystal structures. However, this accuracy has
not been routinely achieved because of limitations in theory and
difficulty with the required calculations. Also, there is a limit to
which similar bond lengths can be resolved, which is dependent on the
data range and is typically of the order of 0.1 Å. Significant
improvements in EXAFS data analysis have occurred in recent years
including the use of constrained and restrained refinement (22-25),
mapping techniques (25, 26), use of three-dimensional crystal structure coordinates (27-29), and full (i.e. including interligand
as well as intraligand) multiple scattering (30, 31). These methods have been combined in the analysis program EXCURV98 (32) and have recently been applied to the copper protein azurin (33) where it
gave rise to a significant improvement in the quality of the final
simulation and model. We apply these methods here to both the
(H)PO4 and (H)CO3 adducts of Fe·nFBP. These
studies are important for understanding the mechanisms for iron
transport in this pathogenic bacterium.
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EXPERIMENTAL PROCEDURES |
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Materials-- Electrophoresis was carried out using a Bio-Rad Protean II Minigel system (protein) and Invitrogen H5 system (DNA). An Amersham Biosciences fast protein liquid chromatography system and columns were used for chromatographic separations of proteins. Pre-cast SDS-PAGE gels (10% bis-Tris) were purchased from Novex and were used according to the manufacturer's instructions.
Monosodium citrate (Aldrich), NaHCO3 (Aldrich), nitrilotriacetic acid (H3NTA, Aldrich), NaH2PO4, and Na2HPO4 (BDH), tetrasodium pyrophosphate (Strem), Hepes (Aldrich), Tris (Aldrich), cetyltrimethylammonium bromide (CTAB, Aldrich), and NaH13CO3 (MSD isotopes, >99% enriched) were used as received. Iron, phosphorus, gallium, bismuth, and cobalt atomic absorption standard solutions were purchased from Aldrich. All other chemicals were reagent grade. A freshly prepared physiological buffer was used with final concentrations of 4 mM NaH2PO4, 100 mM NaCl, and 25 mM NaHCO3 (pH 7.4).
Stock solutions of [Fe(III)(NTA)2], [Ga(III)(NTA)2], and [Bi(III)(NTA)] were prepared from the iron, gallium, and bismuth atomic absorption standard solutions and the stoichiometric amount of H3NTA. The pH values were raised slowly to ~5.5 with microliter amounts of NaOH (1 M). Diluted solutions were used where necessary. Aqua Co(III) ions are unstable under normal conditions, and so Na3Co(III)(CO3)3 was prepared according to published procedures (34, 35). The purity was confirmed by carbon and cobalt analysis. A stock solution of Na3Co(III)(CO3)3 was prepared in 1 M NaHCO3 according to published methods (36). This complex has been used successfully to load human transferrin with Co(III) (36).
Overexpression of nFBP--
nFBP was overexpressed in E. coli Top10 One ShotTM or DH5F' cells (Invitrogen)
transformed with pTRC99A/FBP. A single colony from freshly transformed
cells was used to inoculate 5 ml of 2YT broth
(Bacto-tryptone (16 g), Bacto-yeast extract (10 g), NaCl (5 g)
dissolved in distilled water (1 liter), and then adjusted to pH 7.5 with NaOH) containing 100 µg/ml ampicillin in a sterile 10-ml vial.
This culture was shaken overnight at 310 K and used to inoculate 1 liter of 2YT with 100 µg/ml ampicillin in sterile 500-ml flasks.
After the flask was shaken overnight at 310 K, a pink pellet of cells
was isolated by centrifugation at 10,000 × g for 15 min at 277 K and stored at 253 K.
Purification of nFBP-- nFBP was purified by a modification of the method of Berish et al. (20). The pink pellet (~5 g) was defrosted at room temperature and resuspended in 50 ml of 1 M Tris (pH 8.0). 50 ml of 2% CTAB then was added followed by stirring slowly overnight at 310 K. The white insoluble material was removed by centrifugation at 10,000 × g for 15 min at 277 K. The supernatant (cell-free extract) was diluted to a final volume of 1 liter by adding distilled water and then filtered using Whatman number 4 paper (Fisher). The cell-free extract was applied to a RESOURCE®S cation exchange column (1 or 6 ml, Amersham Biosciences) connected to a fast protein liquid chromatography system, washed according to the manufacturer's instructions, and equilibrated with 10 mM Tris buffer (pH 8.0). The filtered CTAB extract was loaded onto the column at a flow rate of 4 ml/min. Unbound proteins were removed by extensive washing with low salt buffer. Proteins were eluted with a linear NaCl gradient of low-to-high salt (0-1 M NaCl) over 20 column volumes in 10 mM Tris buffer. Pink fractions were collected and were analyzed by SDS-PAGE. nFBP was desalted by dialysis and concentrated by ultrafiltration (Amicon concentrator). The concentration of purified protein was determined by the BCA method using bovine serum albumin as a reference (37).
Preparation of Apo-nFBP-- Apo-nFBP was prepared by treatment of holo-nFBP solutions in a Centricon 10 microconcentrator (Amicon) with 250 mM sodium citrate (pH 4.5) at least four times until there was negligible absorbance at 481 nm. The apo-nFBP was then washed four times with 2 ml of 0.1 M KCl. At this point, the colorless protein was exhaustively exchanged into the buffer to be used in different experiments (Centricon 10).
Mass Spectrometry-- Electrospray mass spectrometry was performed on a Micromass Platform II quadrupole mass spectrometer equipped with an electrospray ion source. The purified holo-protein or apoprotein samples in 10 mM Hepes (pH 7.2) were diluted with MeOH/H2O (1:1, v/v) to a final concentration of 20 µM (20 pmol/µl), and CH3COOH (final concentration 0.5%) was added to the samples before measurement. The final pH of the protein samples was ~3.7. The mass spectrometer was scanned at intervals of 0.1 s, the scans were accumulated, the spectra were combined, and average molecular mass was determined using the MaxEnt and Transform algorithms of massLynx software.
UV-visible Spectroscopy-- All of the UV experiments were performed with 1-cm cuvettes on a computer-controlled Shimadzu UV-1000 spectrometer with temperature control at 298 or 310 K. UV-difference spectra after the addition of metal complex to apo-FBP were recorded immediately and at different time intervals. For titration experiments, aliquots of metal complex (0.5-10 µl) were added and the solution was left to equilibrate at 298 K for 30 min. The binding or release of Fe(III) was monitored by the increase or decrease in absorbance at 481 nm.
NMR Spectroscopy-- FBP samples were dissolved in 0.1 M KCl in 10% D2O, 90% H2O. The pH was adjusted to 7.4 ± 0.1 when necessary using NaOH and HCl (0.1 M). The pH* values (meter readings) of NMR solutions were recorded before and after NMR measurements.
1H NMR spectra were recorded on a Bruker DMX 500 spectrometer at 500 MHz, using 0.6 ml of FBP solution (~1 mM) in 5-mm tubes at 298 K using ~1,000 transients, 6-µs (50o) pulses, 1.8-s recycle time, 16,384 data points, and water suppression via presaturation. The chemical shift reference for 1H was sodium trimethylsilyl-d4-propionate (TSP). Resolution enhancement of the spectra was achieved by processing the free induction decays with a combination of unshifted sine-bell and exponential functions (line broadening of 1.5-20 Hz) on a Silicon Graphics computer using XWIN-NMR software.
Proton-decoupled 31P NMR spectra were recorded on a Bruker DMX 500 spectrometer operating at 202 MHz. Typically, 3,000-10,000 transients were collected using a pulse-width of 6 µs (50o), relaxation delay of 2 s, and 16,384 data points. The 31P reference was external 85% H3PO4. The spectra were processed using exponential functions (line broadening of 2-10 Hz).
Two-dimensional [1H,1H], NOESY (mixing time of 100 ms), and TOCSY spectra (mixing time of 50 ms) (38, 39) were acquired on a Bruker DMX 500 spectrometer at 298 K using 4,096 data points in the f2 dimension [1H], 32-64 increments of t1, acquisition time of 0.2 s, 96 scans, and 512 increments in the f1 dimension in a total time of ~21 h. Qsine functions were used in both dimensions for processing data using XWIN-NMR 2.0 software. The residual water signal was suppressed by presaturation or pulsed-field gradients. Peaks were referenced to TSP.
ICP-AES-- ICP-AES was performed on a Thermo Jarrell Ash IRIS spectrometer using standard methods. Metal-loaded proteins and apoproteins were purified by using Centricon 10 (Amicon) ultrafilters and washing three times with ultrapure water followed by ultrafiltration after each washing. The final protein solution was diluted with Tris buffer (10 mM).
pH Measurements-- The pH values of the solutions were adjusted with HCl or NaOH (DCl or NaOD for samples in D2O solution) and determined using a Corning 240 pH meter equipped with an Aldrich microcombination electrode calibrated with Aldrich buffer solutions at pH 4, 7, and 10. The pH meter readings for D2O solutions are recorded as pH* values, i.e. uncorrected for the effect of deuterium.
X-ray Absorption Spectroscopy-- X-ray spectra were recorded at the iron K edge on EXAFS station 9.3 at Daresbury Laboratory Synchrotron Radiation Source (operating at 2 GeV) using a Si<220> double crystal monochromator and vertically focusing mirror for harmonic rejection. Data for native nFBP and apo-nFBP loaded with Fe(III) in the presence of bicarbonate rather than phosphate were collected at 13 K (using a liquid helium cryostat) in fluorescence mode using a 13-element solid state germanium detector. Data were collected in k space using a k3-weighted regime for counting time with a total scan time of 40 min. 28 scans were collected from each sample. The edge positions were calibrated against an Fe foil. Samples were prepared as follows. Purified native nFBP was concentrated to 7 mM by ultrafiltration and washed three times with 0.1 M KCl. Fe·CO3·nFBP (2 mM) was prepared by reacting apo-nFBP with 1.2 mol eq of Fe(NTA)2 in Hepes buffer (pH 7.4) in the presence of 10 mM sodium bicarbonate. The excess of Fe(NTA)2 and free NTA was removed from the yellowish-pink solution by ultrafiltration washing four times with 0.1 M KCl. Data were processed using EXCALIB and SPLINE (modified for use with EXCURV) (40). The EXAFS data were converted into k space. The photoelectron wave vector k is related to the incident photon energy (E) and to the threshold energy (Eo) by Equation 1,
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(Eq. 1) |
The EXAFS analysis proceeded in a number of stages. (i) Single
scattering analysis of the first shell (ligand or nearest neighbor) atoms was the first stage. The Fe-PO4-nFBP data were
then analyzed as follows. (ii) Constrained (rigid body) EXAFS was fit
using the crystallographic coordinates of hFBP (1.6 Å resolution, PDB code 1MRP) as starting model. The EXAFS data were simulated retaining
the three-dimensional coordinates with full (i.e. including interligand in addition to intraligand) multiple scattering. Protein residues were truncated, keeping only those atoms within 5 Å of central iron atom that should be relatively well ordered with respect
to iron such as all atoms of the phenolate rings of Tyr-195 and Tyr-196
(n = 2 × 7), all atoms of the imidazole
ring of His-9 (n = 5), all atoms of the phosphate group
(n = 5), the terminal part of Glu-57 (n = 4) (CG, CD, OE1, OE2), and water (n = 1). This gave a
total of 29 atoms. Treating each residue as a rigid body means that the
position of all the atoms in a multi-atom ligand can be defined using
only three parameters: one distance (usually the ligand atom) and two
polar angles ( and
) while simultaneously maintaining the
overdeterminancy of the refinement (see Table III) and maintaining the
internal geometry of the residue. (iii) One parameter (Fermi energy)
was refined, and Debye-Waller factors were set at values from final
simulation. (iv) 13 parameters were refined: Fermi energy, 6 distances
for ligand atoms, 5 angles for non-water residues, one single
Debye-Waller factor for the 6 six-liganding (nearest neighbor) atoms.
It was assumed that the Debye-Waller factors increased with distance
from the central atom as it is physically unrealistic to allow more
distant non-liganding atoms to have stronger thermal correlations with
the metal atom than the nearer atoms (23, 25, 46). Debye-Waller factors were grouped together for similar distances and atom type and scaled to
the refined Debye-Waller factor for the ligand atoms. (v) 14 parameters
were refined as described above plus a rotation around the vector
defined by CG-CD of Glu-57. The starting parameters for the rotation
around the CG-CD vector were established by mapping this parameter
against the related positional parameters for the Glu-57 ligand. The
validity of this methodology was tested using the Fe-EHPG data (see
"Results"). (vi) Similar ligand distances (
< 0.1 Å) were
grouped, giving three rather than six ligand distances, and the
refinement was repeated. (vii) Restrained refinement against
"ideal" bond lengths was derived from the Engh and Huber library
(47). A total of 55 two-dimensional 1-2, 1-3, and 1-4 bond length
restraints within residues were applied (23). These restraints
together with not refining
values are sufficient to maintain the
planarity of the phenolate and imidazole rings. A weighting of
EXAFS:restraints of 0.5:0.5 was used. Further details of the procedure
used were described previously (for details see Refs. 23, 33, and
46).
Uncertainties were estimated in two ways; 1) by varying a parameter
away from its optimal value while optimizing all other parameters until
(D)2 (as defined in Equation 1 of Ref. 48) increased by
1 above its minimum value (48) and 2) by calculation of the co-variance matrix. The statistical component of the noise was quantified by
subtracting a smooth function obtained from a low pass Fourier filter
from the background-subtracted data.
Once a satisfactory simulation of the EXAFS data for
Fe-PO4-nFBP had been obtained, PO4 was replaced
with CO3 (giving 28 shells), and the analysis was repeated
with the Fe·CO3·nFBP data. After completion of most of
the EXAFS analysis, the crystal structure coordinates of nFBP (PDB code
1D9Y) at moderate (2.2 Å) resolution became available
(www.rcsb.org).2 They
have been included in the comparisons.
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RESULTS |
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Overexpression and Characterization of nFBP
Cloning and Overexpression of nfbp in E. coli--
The vector
pTRC99A/FBP was used for the overexpression of the nfbp
gene. Wild-type nFBP was overexpressed in both Top10 and DH5F'
E. coli cell lines transformed with pTRc99A/FBP. The
formation of a pink pellet was indicative of the overexpression of
nFBP. Expression levels were similar in 2YT for both cell lines, but Top10 cells containing pTRC99A/FBP gave deeper pink pellets and were
used for large scale protein expression. Optimal yields of protein were
obtained by aerobic cell growth in 2YT at 310 K for 28-30 h. From a
1-liter cell culture, ~100-200 mg of purified protein was normally obtained.
Purification of nFBP-- The overexpressed protein was located in the periplasm of E. coli, because nFBP contains an N-terminal signal sequence that directs FBP to the periplasmic space (20). The cationic detergent CTAB was used to prepare a periplasmic extract. The method of Berish et al. (20) (4% CTAB extraction for 1 h at 310 K) still gave a pink pellet after centrifugation. This suggested that the extraction was not complete. After using 2% CTAB with extraction overnight, a white pellet with red supernatant was obtained after centrifugation, indicating that a complete extraction had been achieved.
The diluted cell-free extract was applied to a cation exchange column that was then eluted with a 1 M NaCl gradient. Pink fractions were eluted at 0.16 M salt and were collected. After dialysis and ultrafiltration, pink-colored high purity nFBP was obtained as shown by the SDS-PAGE gel, which had one major band at ~34 kDa (see supplemental Fig. S1 under "Supporting Information"). These profiles are similar to the FBP purified from gonococci by Mietzner et al. (18). Crystallization trials were successful and confirmed the high purity of the protein.
Characterization of nFBP-- The molecular masses of both holo-nFBP and apo-nFBP were investigated by electrospray mass spectrometry. When the pink holo-nFBP solution was added into the mass spectrometry buffer containing 50% organic solvent (CH3CN or CH3OH), a red precipitate formed that dissolved after gentle shaking, giving rise to a colorless solution. However, no precipitate formed with the apo-nFBP sample. Holo-FBP had an apparent molecular mass of 33,643 Da (see supplemental Fig. S2 under "Supporting Information"), in agreement with the amino acid sequence (309 amino acids without iron or phosphate; theoretical mass of 33,639.39 Da). Apo-nFBP itself had an apparent molecular mass of 33,648 Da. This finding suggests that under the conditions of mass spectrometry neither holo-nFBP nor apo-nFBP had iron or synergistic anion bound to the protein and that the nFBP signal sequence had been cleaved at Asp-23 upon translocation to the periplasm.
The iron and phosphorus contents of both holo-nFBP and apo-nFBP were
determined by ICP-AES. The protein concentrations (holo-nFBP, 5.83 mg/ml; apo-nFBP, 5.64 mg/ml) were measured by the BCA method using
bovine serum albumin as a standard (37) and were converted into molar
concentrations (holo-nFBP, 1.72 × 104
M; apo-nFBP, 1.67 × 10
4 M)
according to their calculated molecular masses. The samples were
diluted 10 times for ICP-AES analysis, and three repeat measurements were made. The iron and phosphorus contents of the proteins are listed
in Table I. These data suggest
that the "as purified" holo-nFBP contains 1 mol iron and 1 mol
phosphorus (as the synergistic anion phosphate)/mol protein, whereas
apo-nFBP contains neither iron nor phosphorus.
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UV-visible Studies--
UV-visible spectra of holo-FBP and apo-FBP
are shown in Fig. 1. Both holo-nFBP and
apo-nFBP exhibit a band with a maximum intensity at 279 nm and a
shoulder near 290 nm, indicative of the presence of Trp residues. The
molar extinction coefficients at 280 nm (280) were
determined as 44,200 M
1cm
1 for
apo-nFBP and 48,900 M
1cm
1 for
holo-nFBP. Apo-nFBP has no band in the visible region, whereas holo-nFBP exhibits a broad band centered near 481 nm, which gives rise
to the pink color of the protein. The value of
481 for
holo-nFBP was determined to be 2,440 M
1cm
1.
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NMR Spectroscopic Studies-- Holo-nFBP was highly soluble in 0.1 M KCl (>7 mM), and the solution was stable at 277 K as well as at room temperature. However, apo-nFBP was much less soluble (<2 mM) and less stable at 277 K but more stable at room temperature. Precipitates formed in apo-nFBP solutions a few days after preparation, even at 0.2 mM.
31P NMR investigations revealed no 31P signal
for either apo-nFBP or holo-nFBP. This was expected for apo-nFBP,
because ICP-AES studies revealed no phosphorus associated with the
apoprotein. However, ICP-AES detected one phosphorus in the
holo-protein, so the absence of a 31P NMR signal suggests
that this phosphorus atom must be close to the paramagnetic Fe(III)
center, which broadens the 31P signal beyond detection.
High spin 3d6 Fe(III) is known to be an effective
NMR-broadening agent. The effects can arise from both through-space
(dipolar broadening and 1/r6) and
through-bond (contract interaction, delocalization of unpaired electrons) interactions (49).
1H NMR spectra for both holo-nFBP and apo-nFBP are shown in
supplemental Fig. S3 under "Supporting Information." Major
1H NMR differences exist between the apo-protein and the
holo-forms. For example, sharp peaks at = 7.78 (a),
= 7.73 (b), and
= 7.24 (c) for
apo-nFBP (as shown in supplemental Fig. S3) have disappeared from the
spectrum of holo-nFBP and are probably attributed to aromatic CH
protons from the residues close to Fe(III). The peaks at
=
0.03 (d),
=
0.60 (e), and
=
0.65 (f) in the high field region are probably from methyl
groups lying below or above side chain aromatic rings (e.g.
Trp) and are shifted by ring current effects. These peaks are broadened
for holo-nFBP, suggesting that they are near to the Fe(III) or are less
mobile in the holo-protein. The two-dimensional TOCSY and
two-dimensional NOESY spectra of apo-FBP and holo-FBP contain many
overlapping peaks because of the large size of the protein (50). More
NOESY connectivities were seen for apo-nFBP than for holo-nFBP,
especially in the region between 5 and 6.6 ppm (50). All of these
features confirm that recombinant apo-FBP and holo-FBP are folded
proteins. Further analysis of the NMR data is beyond the scope of this paper.
Anion Binding Properties of nFBP
We investigated whether nFBP from N. gonorrhoeae contains phosphate as a synergistic anion and whether this can be replaced by carbonate, the normal synergistic anion for transferrin, or by other anions.
UV-visible Studies--
A titration of Fe(III) (as
Fe(NTA)2) with apo-nFBP in the presence of 5 mM
phosphate (10 mM Hepes buffer (pH 7.4)) was monitored by
UV-visible spectroscopy. As shown in Fig.
2A, a new broad band centered
at 481 nm appeared immediately and increased in intensity upon the
addition of Fe(III) until 1 mol eq was reached. A plot of
A481 against the molar ratio (r)
of Fe to apo-FBP is shown in Fig. 2B. It can be clearly seen
that with the increase in r, the band at 481 nm increased in
intensity and reached a plateau at r = 1, which
suggests that one mol Fe(III) binds strongly per mol nFBP.
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Similar experiments were performed using other anions (pyrophosphate,
(bi)carbonate, oxalate, and NTA) instead of phosphate. In all of these
cases, Fe(III) bound strongly to nFBP with a 1:1 molar ratio. A
distinctive position of the LMCT (TyrFe(III)) band was
observed as shown in Fig. 3 and Table
II. The wavelength of the LMCT band of
transferrin is known to be sensitive to the nature of the anion
including the substitution of carbonate by glyoxylate or glycine (51,
52). This finding suggests that both carboxylates and phosphates can
act as synergistic anions for nFBP. The wavelength of the LMCT band in
the presence of phosphate is the same as the band for the native
protein (481 nm), suggesting that phosphate is the native synergistic
anion. A titration performed in physiological buffer (100 mM NaCl, 4 mM phosphate, and 25 mM bicarbonate (pH 7.4)) gave rise to a LMCT band at 481 nm, which suggests that phosphate is the bound anion under these conditions. Phosphate binding was also confirmed by the anion exchange studies described below.
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A titration of Fe(NTA)2 as the Fe(III) source with apo-nFBP
in 0.1 M KCl but in the absence of any added synergistic
anion gave rise to a specific band at 457 nm. This implies that NTA can
also act as a synergistic anion. NTA is known to be able to act as a
synergistic anion in transferrins (51, 52), confirmed by the recent 2.1 Å resolution x-ray crystal structure of an Fe(III)-ovotransferrin-NTA ternary complex (53). The wavelength of the LMCT band of
Fe(III)-NTA-hTF (465 nm) is the same as that for
Fe(III)·CO3-hTF (52). Therefore, it is reasonable that
max for the LMCT band of Fe(III)-NTA-nFBP is similar to
that for Fe(III)·CO3·nFBP (~459 nm).
Because Fe(III) can be loaded into hTF by the addition of Fe(II) to
air-saturated solutions of the apo-protein (54), we titrated apo-nFBP
with Fe(II) ammonium sulfate in the absence of other added anions. The
oxidation of Fe(II) to Fe(III) appeared to be accelerated by the
presence of nFBP and led to a weak Fe·nFBP complex with a
max of 472 nm. The saturation of nFBP was complete after
the addition of ~3 mol eq of Fe(II) (as judged by the ratio A470/A400). Because this
is not the wavelength of the Fe·CO3·nFBP complex, we
conclude that ambient bicarbonate (~0.14 mM (pH 7.4), 298 K (55)) is not able to promote the formation of an
Fe(III)·CO3·nFBP complex. The identity of the bound
anion(s) in Fe·nFBP formed via Fe(II) oxidation is unknown but could
be hydroxide, and it is possible that such a complex is unstable via
the loss of iron as ferric hydroxide. The experiments described below
appear to rule out sulfate as a bound anion.
Titrations of Fe(NTA)2 with apo-nFBP were performed in the
presence of sulfate or perchlorate to see whether they can serve as
synergistic anions. LMCT bands centered at ~458 nm were observed but
were ascribed to bound NTA (458 ~2,800
M
1cm
1) as the synergistic
anion. Moreover, phosphate exchange studies on Fe-PO4-nFBP
monitored by 31P NMR (described below) also do not support
sulfate or perchlorate as a synergistic anion for Fe·nFBP.
Synergistic Anion Exchange
UV-visible Studies--
Fe·CO3·nFBP was prepared
by mixing 1.0 mol eq of Fe(NTA)2 with apo-nFBP (83 µM) in the presence of a large excess of
HCO
|
UV-visible studies were also performed to see whether phosphate in
native nFBP can be displaced by carbonate. As shown in Fig.
5A, when a 200-fold molar
excess of NaHCO3 was added to native nFBP solution (0.11 mM, 0.1 M KCl (pH 8.0)), the LMCT band of native FBP gradually shifted from 481 to 460 nm and increased in
intensity over a period of 5 h. This finding suggests that phosphate in nFBP has been displaced and that
Fe·CO3·nFBP has formed.
|
Similar experiments were also performed using a 400-fold molar excess of sulfate or perchlorate instead of bicarbonate; however, no shifts of the LMCT band of the native nFBP were observed.
31P NMR Studies-- To confirm that the synergistic anion (hydrogen) phosphate can be displaced by (bi)carbonate, 31P NMR studies were carried out. When a 50-fold molar excess of NaHCO3 (pH 8.0) was added to a solution of native holo-nFBP (1 mM (pH 8.0), 0.1 M KCl), no 31P NMR peak appeared after incubation for 12 h at 298 K, which suggests that phosphate binds strongly to Fe·nFBP. However, when a 200-fold molar excess of NaHCO3 was added, a new 31P NMR peak appeared at 3.23 ppm and grew to its maximum intensity 24 h later (see Fig. 5B). This peak has a chemical shift characteristic of free phosphate (56, 57). This confirms that phosphate is the native synergistic anion in nFBP and that it can be displaced by a large excess of carbonate. To confirm that phosphate was displaced, the sample was ultrafiltered. A colorless filtrate was obtained, which gave a 31P NMR peak at 3.23 ppm (i.e. free phosphate).
In contrast, after the addition of a 400-fold molar excess of Na2SO4 or NaClO4 to solutions of holo-nFBP and incubation for 3d at 298 K, no 31P NMR peak appeared. This together with the UV studies suggests that neither sulfate nor perchlorate can act as a synergistic anion for Fe·nFBP.
EXAFS Comparison of Phosphate-Fe·nFBP and Carbonate-Fe·nFBP
FeK-edge EXAFS studies were carried out to investigate the local
structural differences between Fe·PO4·nFBP and
Fe·CO3·nFBP. The edge structure (XANES) and edge
positions were the same for Fe·PO4·nFBP and
Fe·CO3·nFBP, and their positions are consistent with
the expected 3+ oxidation state for Fe. However, the spectra differ
markedly in other respects (see Fig. 6).
The amplitude of the EXAFS oscillations for Fe·PO4·nFBP
dies away with increasing k, whereas the amplitude for the
Fe·CO3·nFBP EXAFS is relatively even throughout its
range. This results in significant differences in the corresponding
Fourier transforms, which are not the result of the different EXAFS
data ranges available for the calculation of the Fourier transforms
(see supplemental Fig. S4 under "Supporting Information"). The
Fourier transform for Fe·PO4·nFBP shows two overlapping
peaks of similar intensity, whereas that for
Fe·CO3·nFBP is dominated by a single peak with a
shoulder on the longer distance side. The two overlapping peaks for
Fe·PO4·nFBP can be simulated with two oxygen
backscatterers at an average distance of 1.94 Å and four
oxygen/nitrogen backscatterers at an average distance of 2.12 Å. The
fit is slightly improved if the nitrogen is allowed to refine to a
separate (2.24 Å) distance. The EXAFS for Fe·CO3·nFBP refines to four donor ligands at an average distance of 1.98 Å and two
ligands at 2.20 Å (the shoulder in the Fourier transform). These peaks
are the result of scattering from the atoms directly coordinated to
Fe(III). The first shell peak in the Fourier transform is considerably
more intense for the carbonate adduct than the phosphate adduct. This
is reflected in the corresponding Debye-Waller factors
(22 = 0.001 and 0.004, respectively). This is not an
artifact of data collection. Both data sets were collected at the same
temperature, and the total count rate on the fluorescence detector was
kept approximately the same for both samples (half of the maximum
linear count rate). This finding suggests that the ligands are
significantly more ordered with respect to the metal with
CO3 bound compared with PO4.
|
Despite the difference between the spectra, both data sets have a peak in their respective Fourier transforms at ~2.6 Å for which the crystal structures have no obvious candidate atoms. The Fe·PO4·nFBP data also have a peak at 3.2 Å, which is at the correct distance for the phosphorus of the synergistic phosphate ligand. Although these data together with the significant difference in the first shell appear to confirm that the two nFBP samples have different synergistic anions, it must be remembered that in addition to small "rigid" ligands such as phosphate and carbonate, the atoms of the Tyr and His ligands contribute significant multiple scattering in the 3-6 Å range. Because the ligand bond lengths are different between the two samples, the Tyr and His multiple scattering patterns will also differ. Thus, it is the different distribution of ligand bond lengths and pattern of peaks in the 3-6 Å range that supports the notion of different synergistic anions. To confirm this finding, the constrained and restrained full multiple scattering analyses in three-dimensional space were carried out using all the well ordered atoms of the ligand residues within 5 Å of the central atom.
Multiple Scattering EXAFS Analysis
EXAFS simulations of the Fe·PO4·FBP data using the
hFBP and nFBP crystallographic coordinates gave very poor results
(R-factors of 60.9% and 73.4%, respectively) (see
supplemental Fig. S5). Given the difference in the estimated errors
between the two techniques (~0.1-0.2 Å for the crystal structures;
~0.02 Å for EXAFS), this is not particularly surprising. A
comparison of the two sets of crystallographic coordinates (see Table
IV) reveals significant disagreement in the Fe-ligand bond lengths. The
nFBP structure gives both Fe-Tyr bond lengths as similar (1.84 and 1.88 Å), whereas the hFBP structure suggests a large split (0.14 Å) with one very short Fe-tyrosine bond (1.78 Å). The two structures
also differ by 0.21 Å in the Fe-OH2 bond length. The
Fe·PO4·nFBP EXAFS data do not support the short Fe-Tyr
distance of the hFBP crystal structure. With the hFBP coordinates, the
R-factor can be reduced from 61 to 42% by changing the
Fe-Tyr-195 bond length from 1.78 to 1.9 Å, which is similar to the
Fe-Tyr-196 distance and more typical of iron-tyrosinate systems. On
(constrained) refinement of the Fe-ligand bond lengths (using either
coordinates), the Fe-OH2 distance refines to 2.10 Å,
slightly longer than the 2.02 Å of the hFBP crystal structure but
still significantly shorter (by 0.13 Å) than the distance suggested by
the nFBP crystal structure. For the constrained refinement, a total of
13 parameters were initially varied: the Fermi energy, 6 Fe-ligand bond
lengths, 5 Fe-ligand angles, and the Debye-Waller factor for
ligand-bonded (first shell) atoms. At this stage, the best fit
obtainable had an R-factor of 30.5% and did not adequately
simulate the peak at 2.6 Å in the Fourier transform or the EXAFS data
between 7 and 9 Å1. Because the 2.6 Å peak is also
present in the Fe·CO3·nFBP data, it was concluded that
it was not likely to be attributable to the synergistic phosphate
anion. There is no independent evidence to suggest an additional
unidentified ligand at this distance. To check whether the assignment
of the ligands to the six refined distances were correct, the ligands
were systematically swapped to the other five distances, and the
refinement was repeated. This finding revealed a number of false minima
with R-factors of 33-35%, but none improved upon the fit
to the 2.6 Å peak. Therefore, it was necessary to consider what more
significant changes from the crystal structure might be responsible for
this difference. The simulation of the "outer" multiple scattering
peaks (3-5 Å) was good. 3-5 Å peaks result largely from the
multiple scattering from the Tyr and His rings. Therefore, attempts to
model the 2.6 Å peak by decreasing the Fe-ligand angles for the Tyr
and His rings all resulted in significantly worse fits. Attempts were made to model a "bidentate" phosphate by (a) removing
the water and rotating the phosphate group to place the oxygen closest
to the water at this position and (b) rotating the phosphate
group to place an oxygen in the 2.6 Å Fourier transform peak. In both cases, this resulted in high R-factors and very poor fits to
the EXAFS, partly because of the easily identified P-atom being at the
wrong distance. Clearly, as a "single atom" ligand, the
OH2 cannot make any contribution to the 2.6 Å peak. This
left Glu-57 as the only possible ligand that could account for the
scattering at 2.6 Å. Additionally, unlike the other ligands, the side
chain of glutamate has significant rotational freedom. The
EXAFS-refined and starting models were examined using the molecular
modeling program XFIT (part of the XtalView package) (58). This
examination suggested that a rotation of the terminal portion of the
side chain of Glu-57 could place atoms at the correct distances to account for the unfilled 2.6 Å peak in the Fourier transform. This
rotation could be achieved in two ways. (i) The same rotamer is
maintained, but the terminal position is rotated around the CG-CD bond
(this maintains the residue at approximately the same position with
respect to the other ligands). (ii) Select the rotamer (using the XFIT
in-built rotamer library) that most closely gives the desired bond
distances to the iron. For method (i), a series of multidimensional
maps of the position of these atoms (still treating the ligand as a
rigid body) against EXAFS Fit Index were calculated. They strongly
supported the notion that such a rotation together with a small
translation (to maintain the correct first shell bond length for Glu)
would lead to a satisfactory fit to the EXAFS data. This
change in the position of Glu-57 immediately led to a
significant reduction in the R-factor from 30 to under 26%
and good fits to both the EXAFS and Fourier transforms. A further
reduction in the R-factor (to 23.9%) was then obtained by
refining the position of Glu-57 together with the original parameters
for the other five ligands, giving the constrained fit in Table III.
The deviation of the CG-CD vector in this model from its initial
position arose during the final constrained refinement of all 14 parameters as the Fe-OE1-Glu angle was refined.
The model derived above was obtained by a left-handed rotation around
the CG-CD vector as orientated in Fig. 7.
This places the Fe-bonded oxygen (OE1) closer to the phosphate ligand
and the longer oxygen (OE2) closer to His-9. Equally valid is the opposite rotation placing Fe-bonded OE1 closer to His-9 and the longer
OE2 closer to the phosphate. On constrained refinement, such a model
gives an acceptable R-factor of 24.8%. Constrained refinement using the alternative rotamer model (as described in (ii)
above) also gave an acceptable fit but with a slightly higher R-factor (25.2%). Because it gave the best fit, only the
model derived by a left-hand rotation of Glu-57 around the CG-CD vector was analyzed further.
|
Restrained refinement against ideal bond lengths (47) led to a further reduction in the R-factor (from 23.9 to 20.5%) without any significant changes to the model (see Tables III and IV and Fig. 7). The restrained refinement was well determined with a parameter/observation ratio of 2.0.
|
|
It was necessary to keep the six ligand distances separate until the final stages of the refinement to accommodate the changes in Fe-ligand distances from the initial model. This also allowed for the comparison with the crystallographic distances. An examination of the EXAFS-derived distances for the Fe-PO4-FBP data shows these distances as falling into three distinct groups: 1) Tyr-195 and Glu-57 (1.93 and 1.95 Å, distance correlation 0.8), 2) Tyr-196, water, and phosphate (2.08, 2.10, and 2.13 Å, distance correlation 0.7-0.8), and 3) His-9 (2.28 Å). The distance correlations among these three groups were low (0.05-0.2). Therefore, the effects on both the constrained and restrained models of allowing only three Fe-ligand distances were examined. This resulted in marginally higher final fit indices and R-factors with distances as expected at average values with respect to the six-distance refinement (see Tables III and IV). In the restrained refinement, some of the effect of this averaging is accommodated by a small increase in the deviation from ideal geometry. Relaxing the bond length restraints resulted in unrealistic and unacceptable deviations from the known internal geometry of the ligands. Also, it did not lead to the simulation of the 2.6 Å peak.
The nFBP crystal coordinates suggest a similar distance for the two tyrosine ligands, whereas the EXAFS data for Fe-PO4-FBP suggest a large split (0.15-0.17 Å). This split is similar to that in the hFBP coordinates, but both tyrosines are displaced to longer distance by ~0.15 Å. Attempts to model the similar Fe-tyrosine distances of the nFBP coordinates in the Fe-PO4-FBP EXAFS data (using both the original and EXAFS-derived rotation of Glu-57 with Tyr-196 and Glu-57 distances swapped) resulted in very poor fits and high R-factors, even after extensive refinement.
Fe-EHPG has been widely used as a model for transferrins (59, 60), because it contains 2 amino, 2 phenoxy, and 2 carboxyl groups in a single hexadentate binding site. It is also a useful model for FBPs. EXAFS data for Fe-EHPG were used to test the validity of the method used to fit the Glu residue in the FBP EXAFS data. Starting with the crystal structure (45), the phenyl rings and carboxyl groups were individually tilted away from their initial (correct) positions by up to 60°. The rest of the structure then was refined against the EXAFS data using multiple scattering and restrained refinement,3 and finally, the maps of the "tilt angle" against other refined parameters were calculated. The back atoms of the phenyl rings were too far from the central atom (4.7-5.6 Å) for small angular tilts (<30°) to have any significant effect on the fit. However, the carboxyl group with its carbon at 2.85 Å is a better analogy to the Glu residue in the FBP structure. This proved much more sensitive to angle in all cases, finding the original angular position of the carboxyl group to within 5-10°. This finding supports the case that with this type of multiple ring-containing site, the use of multiple scattering with constraints/restraints does allow the orientation of a carboxyl (or similar) ligand to be determined with confidence.
For the Fe·CO3·nFBP EXAFS data, the PO4 of the model was replaced with an idealized CO3, placing the bonding oxygens of the two ligands at the same positions and the carbon of CO3 along the phosphate (Fe)O-P axis. The carbonate was rotated around the (Fe)O-C axis, and a clear minimum was found. By analogy with the Fe-PO4-FBP EXAFS, attempts to model a bidentate CO3 resulted in high R-factors and poor fits to the EXAFS. The situation and solution with Glu-57 were identical to the Fe-PO4-nFBP case. Also analogous with the Fe-PO4-FBP data were the correlations for the six separate Fe-ligand distances. The groups found were Tyr-195/Tyr-196 (both 1.96 Å), Glu-57/H2O (2.00 Å, 2.06 Å, respectively) and CO3/His-9 (2.19 Å, 2.21 Å, respectively). The correlations in the six-distance refinements suggest that it is safer to model both EXAFS data sets using only three Fe-ligand distances (see Tables III and IV).
An absolute index of the goodness of fit, which takes into account the
degree of overdeterminancy of the EXAFS fit, is given by the reduced
2 function (
)
)2 should be approximately
equal to
, the number of degrees of freedom, and therefore,
(
)
, (
)2 and
(
)
)2 are
approximately equal to
, the fits are acceptable on a statistical basis, and statistical rather than systematic errors dominate (as may
be expected for this type of data). This finding suggests that the
uncertainties given in Table IV adequately represent the uncertainties
of the best-fit parameter values. These uncertainties were calculated
by varying a parameter away from its optimal value while optimizing all
other parameters until (
)2 increased by 1 above its
minimum value (48). Uncertainties calculated by this method were
compared with values obtained using a co-variance matrix. These two
methods should be equivalent and after rounding gave the same estimates
for the uncertainties.
Examination of the resulting models for both phosphate and carbonate anions (Fig. 7) suggests a possible reason why nFBP has the synergistic anion bound in a monodendate mode. In its suggested new position, OE1 of Glu-57 is well placed to form a strong hydrogen bond (2.1 Å, O-H-O angle 113°) to the Fe-bound water at 2.07-2.1 Å. Additionally, Glu-57 may form hydrogen bonds to PO4 and CO3 synergistic anions if they are protonated. This potential hydrogen-bonding network remains intact with both of the other methods discussed for positioning Glu-57. The hydrogen bond lengths and angles vary but are within the normal range. Thus, there appears to be a structural reason why nFBP, unlike transferrin, binds anions in a monodentate mode. The glutamate side chain carboxylate group of nFBP is part of a hydrogen-bonding network, which both locks in the Fe-bound water and stabilizes the synergistic anion in a monodentate configuration.
Metal Binding to nFBP--
Transferrin can bind strongly to metal
ions such as Ga(III), Co(III), and Bi(III) in its specific iron sites
(63). Therefore, investigations were made to determine whether these
metal ions can also bind to nFBP. Metal ion binding to the phenolate
groups of the two tyrosine residues in the specific iron sites of
apo-TF leads to two new absorption bands near 241 and 295 nm because of
the perturbations of the -
* transitions of the aromatic rings (61). This effect has been used to monitor specific metal binding to
transferrins by UV-visible difference spectroscopy (13), and therefore
was used here to investigate the binding of Ga(III), Bi(III), and
Co(III) to apo-nFBP.
Ga(III) Binding--
The addition of Ga(NTA)2 to
apo-nFBP in 10 mM Hepes (pH 7.3) solution containing 5 mM phosphate at 298 K resulted in two new absorption bands
near 245 and 298 nm in the UV difference spectrum. The spectra
are characteristic of metal-tyrosyl coordination and are similar to
those reported for Ga-transferrin complexes (55, 61), suggesting that
Ga(III) binds to the specific iron site in apo-nFBP. A series of
typical UV difference spectra is shown in supplemental Fig.
S6A under "Supporting Information." A plot of
A245 as a function of r, the molar
ratio of total Ga(III) to total FBP, is shown in supplemental Fig.
S6B under "Supporting Information." The non-linear
increase in
A245 for points below r = 1 and the continued increase in
A245 for points beyond r = 1 imply that the binding of Ga(III) to apo-nFBP is rather weak, so that
excess Ga(III) is needed to saturate the site of apo-nFBP.
The weak binding of Ga(NTA)2 to apo-nFBP may be attributed
to NTA competition (Ga(NTA), log KML = 16.20).
To test this possibility, apo-nFBP was titrated with
Ga(NO3)3 (1.43 mM, prepared by
dilution and adding NaOH to gallium atomic absorption standard solution to give pH 2.16) under similar conditions in the absence of any chelating ligands. Ga(III) binding to the specific iron site in apo-nFBP was observed as evidenced by the appearance of two new absorption bands near 245 and 295 nm in the UV difference spectrum (see
Fig. 8). The binding was now stronger as
evidenced by the more intense bands at 245 and 298 nm
(245 = 8,610 M
1cm
1 by r
= 2). This shows that NTA does compete with apo-FBP for Ga(III) binding. However, the increase in
A245 for points below, r = 1 is still non-linear and still continued to increase beyond r = 1. This implies that binding of Ga(III) to apo-nFBP
is not strong. The
values are much lower than those for
Ga2-transferrin (
242 ~34,000
M
1cm
1) and
Ga2-lactoferrin (
242 ~30,000
M
1cm
1) in the absence of
chelating agent (55, 62), implying that the Ga-nFBP binding is weaker
than Ga-transferrin or Ga-lactoferrin binding. Such weak binding in the
absence of any competing ligand is probably related to the hydrolytic
properties of the Ga(III) ion. In aqueous solution near pH 7.4, the
major unchelated gallium species is Ga(OH)
(Ga(OH)
4OH = 10
16.6 (62)) as discussed
previously for Ga-transferrin interactions (55).
|
Co(III) Binding--
Apo-FBP was titrated with
[Co(III)(CO3)3]3 in 10 mM Hepes buffer (pH 7.4) in the presence of 5 mM carbonate or 5 mM phosphate. However, no new
bands appeared in the UV difference spectra. This suggested that
specific binding of Co(III) to apo-nFBP is very weak under these
conditions or too slow to be observed.
Bi(III) Binding--
Titrations of Bi(NTA) with apo-FBP were
performed in 10 mM Hepes (pH 7.3) in the presence of 5 mM phosphate (see supplemental Fig. S7 under "Supporting
Information") or bicarbonate at 298 K. Specific binding was observed
as evidenced by the appearance of two new absorption bands near 244 and
297 nm in the UV difference spectrum. However, as for Ga(III), the much
smaller increase in intensity of the bands at 297 and 244 nm
(244 ~4,060
M
1cm
1 by r = 1)
compared with Bi-hTF(N-lobe) (
241 ~22,000
M
1cm
1 (63)) or
Bi2-hTF (
241 ~21,900
M
1cm
1/Bi (63)) suggests that
binding is rather weak.
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DISCUSSION |
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The successful overexpression of nFBP in E. coli and the subsequent highly efficient purification from a periplasmic extract by cation exchange column chromatography gave a high yield of recombinant nFBP (~100-200 mg from 1 liter of cell culture). Electrospray mass spectra showed essentially the same molecular mass for both holo-nFBP and apo-nFBP (33,643 and 33,648 Da, respectively), which agrees with the predicted molecular mass of 33,639 kDa. This confirms that the nFBP N-terminal signal sequence was recognized, efficiently processed, and cleaved to give a mature protein of 309 amino acids. Also the data show that no metal or synergistic anion was bound to the protein under mass spectrometric conditions, i.e. low pH (<4) in the presence of 50% CH3OH. The release of metal was also supported by the formation of a pink holo-FBP precipitate upon the addition of 50% CH3OH, which subsequently redissolved with the formation of a colorless solution. Holo-nFBP is more soluble than apo-nFBP in aqueous solution, but the opposite is true for solutions containing organic solvents, which suggest that apo-nFBP is more hydrophobic than holo-nFBP. A comparison of the recently determined crystal structure of apo-hFBP (64) with that of holo-FBP (9) reveals that large conformational changes occur upon the removal of the metal ion from the protein. Similar changes are likely to occur for nFBP.
The crystal structure of hFBP (9) suggests that Fe coordinates to phosphate. However, the protein purification process involved washing FBP-containing cells in phosphate-buffered saline, a process that could result in synergistic anion exchange in FBP (2). Also, it is difficult to distinguish phosphate from its isostructural analogue sulfate by x-ray crystallography. In a recent 31P NMR study of FBP (17), FBP-containing cells were again washed with phosphate, and holo-FBP was reconstituted from apo-FBP in the presence of 50 mM phosphate. Also, it was surprising that phosphate bound to paramagnetic Fe(III) gave rise to a resolvable 31P NMR peak. In transferrin, the 13C resonances of bound (13C) carbonate are broadened beyond detection (65). In our holo-FBP sample, the 31P resonance of Fe-bound phosphate also broadened beyond detection. Our data provide unambiguous compelling evidence that phosphate is the synergistic anion in recombinant FBP from E. coli. Our protein purification procedure does not involve any phosphate contamination, and ICP-AES data gave a 1:1:1 molar ratio of P/Fe/FBP in holo-FBP and 0:0:1 ratio in apo-FBP. After adding excess carbonate to holo-FBP solution to displace the bound phosphate, a 31P NMR resonance for free phosphate was detected (Fig. 5B). These findings indicate that phosphate is present as an exogenous ligand, the synergistic anion, and are supported by our UV-visible and EXAFS data.
The pink color of native nFBP or that which develops when Fe(III) is
added to the apo-nFBP solution is attributed to an intense charge-transfer absorption band at 481 nm. The similarity of the visible spectrum to those of transferrins (13) and metal-phenolate complexes (66) suggests that they arise from tyrosine-metal interactions and that the charge-transfer band must be derived from a
ligand phenolate ()
metal (d
*) transition as in the case of
transferrins (67). The molar extinction coefficient at 481 nm
(
481) was determined to be 2440 M
1cm
1, a value close to that
reported by Nowalk et al. (21) and comparable with half that
of Fe2-hTF (
465 = 4950 M
1cm
1 (68)). The molar
extinction coefficients at 280 nm for apo-nFBP and holo-nFBP were
determined as
280 = 44,200 for apo-nFBP and
280 = 48,900 for holo-nFBP, approximately half of those
for apo-hTF and holo-hTF (
280 = 93,000 M
1cm
1 and 103,000 M
1cm
1, respectively (69)).
The wavelength of maximum absorption is affected by the bound synergistic anion, varying from 450 to 481 nm from oxalate to phosphate. Different synergistic anions may affect the energy levels of Fe(III) and cause small changes in the Fe-O (Tyr) bond lengths (Table IV), leading to differences in the energy levels between the charge donor (Tyr) and acceptor (Fe(III)) and contributing to the LMCT bandshifts in UV-visible spectra. Besides phosphate, other anions such as carbonate, oxalate, NTA, and pyrophosphate can serve as synergistic anions for Fe(III) in nFBP. The failure of phosphate to act as a synergistic anion for transferrins is probably because of its tetrahedral structure that cannot provide the appropriate geometry for interactions in the specific metal-anion-transferrin complex, whereas smaller carboxylates can bind to the metal ion as bidentate anions, leaving no space for the binding of water (13). This could also contribute to the stronger binding of iron (compared with nFBP). nFBP can accommodate both phosphates and carboxylates as synergistic anions, probably because the iron-binding cleft in FBP is larger and nearer to the surface and, therefore, more solvent-accessible. Phosphate can provide more sites than carbonate for hydrogen bonding with the protein side chains in the cleft. This may contribute to the stronger binding compared with carbonate. The state of protonation of bound phosphate or carbonate is unknown. Sulfate and perchlorate, although isostructural with phosphate, fail to act as synergistic anions under the conditions we have tried. This may attributed to their weaker coordination properties and weaker hydrogen-bonding ability arising from the lower polarity of Cl-O and S-O bonds compared with P-O and C-O bonds.
A comparison of the models determined by EXAFS for
Fe·PO4·nFBP and Fe·CO3·nFBP shows that
a series of minor changes in Fe-ligand bond lengths and angles together
with a significant difference in overall backscattering intensity
(reflected in the Debye-Waller factors) are responsible for the
markedly different spectra. Viewed simplistically, Debye-Waller factors
are a measure of the thermal and static disorder with respect to the
Fe(III) atom. As the spectra were collected at the same low temperature
and having ruled out factors during data collection, this difference is
probably because of a real difference in static disorder. The largest
change in distance on changing the synergistic anion from phosphate to
carbonate is the shortening of the Fe-O(Tyr-196) distance by 0.12 Å.
The shorter distance for the carbonate complex can be directly equated with its larger extinction coefficient compared with the phosphate complex. The accuracy of metal-ligand bond lengths in protein x-ray
crystal structures is notoriously difficult to assess unless diffraction data to atomic resolution (better than 1.2 Å) are available. Global estimates vary for lower resolution diffraction but
range from ~0.1 to 0.15 Å for 1.5-2.0 Å diffraction data to 0.2
Å for >2 Å data (70). The x-ray structure of hFBP (9) was determined
at 1.6 Å and nFBP as 2.2 Å, suggesting that bond length errors may be
of the order of 0.1 and 0.2 Å, respectively. Thus, the differences
observed between Fe-ligand bond lengths in the two crystal structures
compared with the Fe-PO4-nFBP EXAFS are mostly within the
estimated errors for each crystal structure. It should be noted that
the differences observed for three of the bond lengths in the hFBP
(Glu-57, 0.13 Å; Tyr-195, 0.15 Å; Tyr-196, 0.16 Å) and one from the
nFBP structure (Tyr-196, 0.20 Å) are at the top end of the range. The
bond length errors from EXAFS do not have this same dependence and have
been calculated to be in the range of <0.01-0.04 Å for liganding
atoms in this study. Thus, this analysis is a good illustration of the
complementarity of x-ray diffraction and EXAFS for metalloprotein
studies. The crystal structure provides the metal site topology and
(approximate) metal-ligand bond lengths, whereas EXAFS "fine tunes"
these bond lengths to the equivalent of atomic resolution. As
crystallographic data are not available for the CO3 adduct,
the EXAFS data are currently the only structural information on FBPs
with an alternative biologically relevant synergistic anion bound.
Whereas EXAFS is capable of determining metal-atom distances with high accuracy, it is not able to measure other inter-atom distances to anywhere near this level with the exception of unusual cases (e.g. simple high symmetry systems with significant multiple scattering). Therefore, it is appropriate to consider the accuracy of the glutamate position and of the glutamate-water and glutamate-anion hydrogen bond lengths. The distances of the individual glutamate atoms to Fe are accurate to the normal level for an EXAFS analysis as direct Fe-atom distances have been determined. The geometry of the fragment has been preserved through the use of constrained (rigid body) and restrained refinement methods. However, the absolute position of the fragment with respect to the other ligands will have significantly greater uncertainty because assumptions had to be made regarding how to achieve the rotation. In placing the glutamate residue, it was assumed that there was no movement of the protein backbone, no change in any bond lengths within the glutamate, and that movement could arise only from an allowed rotation (around the CG-CD bond or CA-CB bond for the alternative rotamer) and a change in the bond angle between the ligand and the metal. Thus, the absolute position of the glutamate is chemically reasonable and is the best fit to the data. Comparisons of the three methods used to position Glu-57 and the crystallographic coordinates suggest an uncertainty in the absolute position of the glutamate with respect to the other ligands of approximately 0.7 Å. Because most of this is attributed to different angular orientations, the effect on the proposed hydrogen-bonding network is smaller than this. Hydrogen-bond lengths and angles are within acceptable values in all three models of the Fe·PO4·nFBP data and also for the Fe·CO3·nFBP data.
The difficulty in fitting the crystallographic coordinates to the EXAFS data and reliability of both the EXAFS data and fit have been extensively explored. Reference data collected at the same time show that the EXAFS data are reliable. Additionally, the 2.6 Å peak is present in two otherwise quite distinct data sets. The difficulty in fitting the 2.6 Å peak would not have been observed if only the first shell (using single scattering) had been fitted. Multiple scattering analysis is not new, but retaining the three-dimensional coordinates is a more recent innovation. However, this difficulty would still have been present without the three-dimensional information (i.e. if multiple scattering only within each ligand had been included). A large number of permutations of the structure was examined, but none involving the tyrosines, histidine, synergistic anion, or water ligands fitted the 2.6 Å peak. The use of constraints and restraints to maintain the internal geometry of the ligands significantly reduces the number of degrees of freedom of the model, thereby increasing the likelihood that a unique solution has been found. An examination of the calculated scattering pathways for the best EXAFS fit using the crystallographic Glu orientation (see Table III, Constrained Fit before rotation of Glu-57) suggests that the absence of calculated scattering at 2.6 Å is not the result of destructive (out of phase) scattering pathways. Two carbon/nitrogen/oxygen atoms were required to fit this peak. Although a fit could have been obtained by including an additional ligand(s) at this distance, there is no evidence to support such a model. However, this peak and the EXAFS data were readily modeled on changing the Glu position. The sensitivity of EXAFS data on this type of structure to the orientation of a carboxylate group is amply demonstrated by the Fe·EHPG complex. The overall similarity of the Fe·PO4·nFBP and Fe·CO3·nFBP EXAFS models and the ease with which the CO3 data were fitted after rotation of the Glu residue add credence to the veracity of the model. The changes to the crystallographic model required for Glu-57 involved an allowed rotation of a very flexible structure. Therefore, the EXAFS model is structurally allowed, chemically plausible, and is by far the best fit to the data. We cannot rule out the possibility that the difference in the position of the side chain of Glu-57 suggested by the EXAFS is a consequence of the difference in the data collection temperature between the EXAFS and the diffraction data. The EXAFS data were collected at 13 K, the nFBP diffraction data were collected at 100 K, and the hFBP diffraction data were collected at room temperature. The collection of protein diffraction data at 100 K has become routine in recent years, and there is a growing literature on the differences observed between protein structures at room temperature and at 100 K. Rotations of flexible side chains, especially of aspartate and glutamate, are fairly common. Collecting protein diffraction data at temperatures close to 13 K is still uncommon, and there is very little literature comparing observed structural differences at these temperatures. The orientation of the side chain of Glu-57 is the same in both crystal structures; therefore, the orientation proposed by EXAFS is not a result of the glass transition. However, we cannot rule out a structural/phase transition between 100 and 13 K (71). Other possible reasons for a real structural difference include pH and the method used to reconstitute the holo-protein. The pH value appears to be an improbable reason, because the pH of the EXAFS samples (pH 7.4) is close to that for the hFBP crystal structure (pH 6.6). A large excess of the synergistic anion was used when preparing the samples. It is conceivable that such a large excess displaced the conformational equilibrium away from the crystallographically observed conformer and toward the EXAFS-observed conformer. It may be possible that both the crystallographic and EXAFS-derived Glu orientations fit the crystallographic electron density maps. Such a scenario could arise if the electron density was not able to distinguish between the two orientations at the resolution available or if the electron density could represent a mixture of the two orientations with only one orientation modeled. Finally, it should be remembered that EXAFS is not readily able to distinguish mixtures. That the single orientation derived from EXAFS could be an average of the crystallographic orientation and some other undetermined orientation cannot be ruled not.
Because Ga(III), Bi(III), and Co(III) bind strongly to hTF, which is
structurally homologous to nFBP, the weak binding of these metal ions
to nFBP is surprising. There was competition between NTA and apo-FBP
when Ga(NTA)2 was used as a Ga source. Whether the binding
constants are of the same order as those for metal-hydroxide binding,
the binding may be greatly affected by the hydroxide concentration in
the solution, i.e. pH. Taboy et al. (17)
determined an effective binding constant for Fe(III) binding to nFBP of
2.4 × 1018 M1 (pH 6.5) (298 K, 0.35 mM phosphate), which is within an order of
magnitude of that for Fe(III) binding to the N-lobe of transferrin in
the presence of carbonate. These differences may be attributed to the
much greater solvent exposure and solvent accessibility of the iron
site in FBP compared with transferrin.
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CONCLUSION |
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We have prepared nFBP in high yield by cloning and overexpression
of nfbp in E. coli. Our ICP-AES and
31P NMR data suggest that phosphate is the native
synergistic anion in recombinant nFBP from E. coli. Bound
phosphate can be displaced by treating the holo-protein with excess
bicarbonate, and EXAFS studies revealed marked changes in the local
structure of Fe-PO4-FBP compared with
Fe·CO3·FBP as a result of bond length and angle changes
together with a significant difference in local ordering. EXAFS data
allowed the refinement of the crystallographically determined
metal-ligand bond lengths and suggested that the water, Glu, and
monodentate synergistic anion are involved in an H-bonding network
around the Fe site. The accurate structure of the Fe/anion site
provides a reliable structural basis for rational design of novel
chemotherapeutic agents targeted on this key protein, which is required
for virulence and iron uptake in certain pathogenic bacteria. Oxalate,
NTA, and pyrophosphate also appeared to be capable of serving as
synergistic anions but not sulfate or perchlorate. Apo-FBP is much less
stable than the holo-protein and binds only weakly to Ga(III) and
Bi(III), unlike transferrin. Therefore, attempts to exploit the FBP
route for periplasmic iron transport using heterometal ions, a possible
strategy for the design of novel metalloantibiotics, will need to be
based on other metals.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. R. Strange (Daresbury Laboratory) for the use of Fe-EHPG EXAFS data and Dr. H. Li (University of California, Irvine) for assistance with handling crystallographic data.
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Note Added in Proof |
---|
Evidence for a phase change between 100 and 15 K has recently been reported for protein crystallographic data (71).
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FOOTNOTES |
---|
* This work was supported in part by an Overseas Research Scholar award from the Committee of Vice-Chancellors and Principals, a scholarship (to M. G.) from the University of Edinburgh, and Showcase and Joint Infrastructure Fund awards from The Wellcome Trust (Edinburgh Protein Interaction Centre).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 on-line version of this article (available at
http://www.jbc.org) contains Figs.
S1-S7.
§ Present address: Dept. of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697.
To whom correspondence should be addressed. Tel.:
44-131-650-4729; Fax: 44-131-650-6452; E-mail:
p.j.sadler@ed.ac.uk.
Published, JBC Papers in Press, October 7, 2002, DOI 10.1074/jbc.M208776200
2 D. E. McRee, C. M. Bruns, P. A. Williams, T. A. Mietzner, and R. Nunn, unpublished data.
3 Distance-based constrained refinement with distance refinement is not appropriate for the EHPG ligand as the constraints will also prevent any metal-ligand distance changes.
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ABBREVIATIONS |
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The abbreviations used are: FBP, ferric ion-binding protein; nFBP, ferric ion-binding protein from N. gonorrhoeae; hFBP, ferric ion-binding protein from Haemophilus influenzae; EXAFS, extended x-ray absorption fine structure; TF, transferrin; hTF, human serum transferrin; CTAB, cetyltrimethylammonium bromide; H3NTA, nitrilotriacetic acid; ICP-AES, inductively coupled plasma atomic emission spectrometry; XANES, x-ray absorption near edge structure; Fe-EHPG, N,N'-ethylene-bis(o-hydroxyphenylglycine)-iron(III); LMCT, ligand-to-metal charge-transfer.
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