(Received for publication, April 4, 1997, and in revised form, June 26, 1997)
From the Department of Chemistry and Biochemistry,
University of Oklahoma, Norman, Oklahoma 73019 and
§ Departamento de Microbiologia, Universidade de São
Paulo, São Paulo 08805-900, Brazil
The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B·FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex.
Like other TonB-dependent outer membrane proteins, FepA serves as a receptor for a metal chelate (ferric enterobactin (FeEnt))1 and for noxious agents (colicin B (ColB) and colicin D (ColD)). Like other outer membrane (OM) porins (1-3), FepA contains a hydrophilic channel (4), but the FepA pore (5) is closed by cell surface loops that impart binding and translocation specificity. Thus FepA is a TonB-dependent, energy-dependent, ligand-gated porin: its surface loops open in response to ligand binding and TonB action, to internalize FeEnt (6).
The siderophore FeEnt and the cytotoxins ColB and ColD differ in size, structure, and uptake mechanism, yet all three require FepA and TonB for passage into the periplasm (7, 8). The fifth proposed surface loop of FepA (PL5), bounded by residues 255-336, interacts with all three ligands during their passage through the OM (9, 10). Deletions in this loop abolish FeEnt transport, render the bacteria ColB- and D-resistant (11), and convert FepA into a general porin through which sugars, antibiotics, and other small molecules may enter the cell (5).
Colicins are tripartite cytotoxins: their central, N- and C-terminal domains participate in ligand binding, translocation, and bacterial killing, respectively (13). ColB and D are homologous in their translocation and binding domains, but divergent in their C termini, consistent with their different killing modes: ColB kills by forming a pore in the Escherichia coli inner membrane, whereas ColD acts as a ribonuclease in the cytoplasm.
Colicins may traverse the OM through porin channels (14). This idea originates in part from experiments on a colicin-resistant mutant of OmpF (G119D) with an occluded channel (15). Colicins A and E1 bind to external loops of OmpF and BtuB, respectively, and contain N-terminal determinants that confer pore specificity (16), suggesting that after adsorption they interact with the underlying transmembrane channels. However, no direct physical evidence exists to prove the passage of colicin polypeptides through OM porins.
We examined the mechanism of ColB binding to FepA in vivo with an 125I-colicin binding assay and in vitro with a flourescent derivative of FepA. In both conditions the siderophore and the colicins showed complex binding kinetics. A rapid phase occurred first, followed by a slower step, and both steps were independent of energy and TonB. FeEnt bound about 10-fold faster than ColB during the second stage. The high affinity binding equilibria between ColB and FepA facilitated purification of the receptor and its mutants, by affinity chromatography with immobilized ColB.
We isolated ColB from E. coli DM1187/pCLB1 (obtained from M. A. McIntosh) and ColD from E. coli CA23 (17). The former strain constitutively expresses ColB, and we induced the latter to ColD overexpression by incubation of mid-log cultures with mitomycin C (2 µg/ml; Sigma) for 2 h in the dark. We purified both toxins by selective precipitations and chromatography (17, 18), using specific killing activity and SDS-PAGE as measures of purity. Bacteria (20 g) were harvested by centrifugation, washed with 100 mM potassium phosphate, pH 7.4, resuspended in 50 ml of 20 mM phosphate buffer, pH 7.4, and lysed by 4 min of sonication in 30-s bursts, with cooling below <10 °C. The cell lysate was clarified by centrifugation at 100,000 × g for 45 min, and nucleic acids were precipitated with 0.5% polyethyleneimine (Polymin P, Sigma) and removed by centrifugation at 7000 × g for 30 min. ColB or ColD in the supernatant was precipitated with ammonium sulfate (between 28 and 55% for ColB, and between 41 and 49% saturation for ColD) and collected by centrifugation at 10,000 × g for 30 min at 4 °C. The pellet was resuspended in, and dialyzed against, 50 mM Tris-Cl, pH 7.4, then chromatographed on DE52 cellulose (Whatman). Colicin was eluted with a 0.0 to 0.5 M gradient of NaCl. Peak fractions of interest were concentrated by ammonium sulfate precipitation (55% for ColB and 49% for ColD) and purified over Sephacryl S-300-HR (110 × 1.0 cm) in the same buffer.
FepA PurificationFepA was purified by differential extraction with Triton X-100 (19) and ion exchange chromatography (20), or by affinity chromatography on ColB-Sepharose, synthesized by coupling ColB to cyanogen bromide (CNBr)-activated Sepharose 4B (21, 22). Crude or partially purified FepA was loaded on the column, which was washed with 10 volumes of TTE buffer (2% Triton X-100 in 50 mM Tris-Cl, 5 mM EDTA, pH 7.4), and FepA was eluted with a gradient of 0 to 2.5 M NaCl in TTE. Fractions were analyzed by SDS-PAGE. We regenerated the column with a reverse urea gradient (6.0 to 0.0 M in TTE).
Rabbits were immunized weekly for 1 month
with purified FepA, emulsified with complete (first immunization) or
incomplete (subsequent immunizations) Freund's adjuvant. Rabbits were
bled in week 5 and thereafter. The IgG fraction of the resulting
-FepA serum was purified and conjugated to CNBr-activated Sepharose 4B (22).
Protein concentrations were
determined by the method of Lowry et al. (23), modified for
accuracy in the presence of Triton X-100 (24, 25). The concentration of
ColB was determined by the absorbance at 280 nm using a molar
extinction coefficient of 62,160 M1
cm
1, calculated from its primary sequence (26).
59Fe-Enterobactin was prepared and purified as described previously (27).
ColB Killing AssaysPurified colicin was serially diluted
in LB broth in microtiter plates, from 101 to 3.2 × 10
9, and transferred with a sterile
CloneMasterTM (Immusine Corp., San Leandro, CA) to an LB
plate containing ampicillin (10 µg/ml), seeded with the tester
strain. Colicin titers were measured after overnight incubation, as the
inverse of the highest dilution that cleared the bacterial lawn.
ColB was iodinated with IODO-BEADs (Pierce). Six beads were rinsed with 500 µl of MOPS-buffered saline (MBS; 40 mM MOPS, 0.9% NaCl, pH 6.9), dried, and incubated with 1.0 mCi of Na125I in 300 µl of MBS for 5 min. 2.8 mg of ColB in 3.9 ml of MBS was added for 10 min at 25 °C. The reaction was stopped by removing the beads, and unreacted 125I was eliminated by chromatography over Sephadex G-50 in MBS.
Fluorescence MeasurementsThe site-directed mutant FepA protein E280C was utilized for introduction of fluorescent probes onto the receptor. This mutant protein has been extensively studied and manifests a wild-type phenotype, even when covalently modified at the E280C site (6, 10, 12, 31). Its structural integrity in vivo was assessed with a battery of tests including accessibility to monoclonal antibodies, expression levels, binding and transport of ferric enterobactin, and binding and killing by colicins B and D. Purified FepAE280C-fluorescein bound ferric enterobactin with a Kd almost identical to the wild type FepA, providing good evidence that the overall structure of E280C-Fl was intact. We cannot completely rule out that fluorescent labeling may have some impact on the binding of ligands, but based on the above studies, we believe it is minimal. FepAE280C-Fl fluorescence was recorded with an SLM 8000C fluorimeter upgraded to 8100 functionality (SLM Instruments, Rochester, NY), equipped with a 450-watt xenon light source and a cooled photomultiplier tube housing and operated in photon-counting mode. The excitation and emission wavelengths were set to 490 and 520 nm, respectively. The ratio F/F0 was calculated for each experimental point by dividing the net fluorescence (F) by the net fluorescence intensity at the beginning of an experiment (F0).
Colicin B Binding AssaysAdsorption of purified 25I-ColB to RWB18-60 (fepA) (11) and RWB18-60/pITS449 (fepA+) (11) was measured at 0 °C. Bacteria were grown to mid-log in minimal MOPS medium, and 2 × 108 cells were suspended in 50 µl of MBS. 10 µl of 125I-ColB (varied from 0.075 to 2.0 µM, final concentration) were added and incubated 10 min, the reaction mixture was diluted to 560 µl, and the cells were pelleted at 14,000 rpm for 2 min and counted in a Beckman gamma counter.
Data AnalysisStatistical analyses were performed by nonlinear least squares methods using Grafit (28). For ColB binding, data were analyzed by the bound versus total equation. For fluorescence titrations, a form of the bound versus total equation was used that accounts for dilution of the fluorescent protein and the ligand due to sequential additions of ligand. Fluorescence time course data were fitted with both single and double exponential decay models, for increasing or decreasing functions.
FepA in
RWB1860/pITS449, cultured under iron stress in MOPS medium, bound FeEnt
with a Kd of 24 ± 8 nM to a
capacity 103,000 molecules/cell (Fig. 1).
The siderophore reached equilibrium with FepA within 1 min. The
affinity of the receptor for colicins was somewhat lower; it bound ColB
with a Kd of 185 ± 45 nM, to a
capacity of 96,000 molecules/cell (Fig. 1). Measurements of FepA
expression in 125I-protein A Western blots (75,000 monomers/cell; data not shown) were consistent with the observed
siderophore and ColB binding capacities, indicating approximately 1:1
stoichiometry for the binding of either ligand to the FepA monomer.
FepA bound ColD, under the same experimental conditions, with a
Kd of 560 ± 130 nM, to a capacity
of 33,000 molecules/cell, 3-fold lower than ColB (Fig. 1). Colicin
binding experiments were incubated from 10 min to 3 h with no
significant change in the parameters (data not shown), indicating that
the reactions were at or near equilibrium.
In Vivo Competition between FeEnt and ColB for Binding to FepA
A 10-min preincubation of bacteria with saturating FeEnt
(8.2 µM), or inclusion of saturating siderophore in the
125I-ColB binding assay, prevented ColB binding (Fig.
2A). These data reiterate that
FeEnt protects E. coli from ColB killing by occupancy of a
common binding site on FepA (29, 30). The 10-fold lower affinity of
ColB for FepA predicted a faster off-rate for the toxin, but if ColB
was added first and excess FeEnt subsequently, almost no colicin
dissociation occurred (Fig. 2B), despite the ability of the
siderophore to exclude toxin binding. This discrepancy suggested an
unaccountably slow dissociation of the ColB·FepA complex, and that
ColB adsorption was effectively irreversible in vivo, even
at 4 °C.
To further study the slow release of ColB from FepA, we used conditions
that prevented its readsorption, saturating FeEnt. We equilibrated
bacteria with 3.5 µM 125I-ColB, then diluted,
pelleted, and resuspended the cells in buffer with 10 µM
FeEnt. From the radioactivity of the pellet, measured at intervals, we
calculated koff for ColB, 4.7 × 105 s
1, a half-life
(t1/2) of 4 h. The rate of 59FeEnt
release under the same conditions was 6.9 × 10
4
s
1, a half-life of about 20 min. While the slow off-rates
explained the ligand competition results, they were inconsistent with a one-step binding mechanism; assuming single-step ColB binding and a
kon of 108
M
1 s
1 (an order of magnitude
slower than the diffusion limit for small molecules), the measured
Kd of 185 nM predicted a dissociation t1/2 of less than 1 s. The slower observed
ligand dissociation rates suggested that the measured
Kd values contained rate constants for more than one
binding step. The simplest explanation was that ColB bound in two
steps, a rapid initial phase and a second phase that locked the
receptor and ligand into a slow dissociating complex.
The mutant protein FepAE280C
(6, 10, 12, 31) binds and transports FeEnt, ColB, and ColD at wild type
levels. Residue 280 lies in a proposed surface loop that participates
in ligand binding (9, 10). We derivatized FepAE280C with the
sulfhydryl-specific reagent
5-iodoacetamidofluorescein.2
The fluorescence intensity of the labeled protein (FepAE280C-Fl) in DM
micelles decreased approximately 40% upon the addition of FeEnt (Fig.
3); analysis of this quenching gave
a Kd of 15 nM, in agreement with values
derived from other measurements of FeEnt binding (10, 20). The
fluorescence intensity of FepAE280C-Fl also decreased upon binding of
ColB, but only about 15%, indicating that the siderophore and toxin
interact differently with the labeled site (Figs. 3 and
4). As seen in vivo, ColB and
FeEnt competed for binding to FepA in vitro; FeEnt excluded
ColB, and vice versa (Fig. 3)
The reduction in FepAE280C-Fl fluorescence that occurred upon binding
of FeEnt or ColB followed a double exponential decay (Fig. 4).
Convergence did not occur with a single exponential decay model.
Fluorescence decreased during FeEnt binding (Fig. 4B) with
an initial rapid phase (k1; 1.8 × 102 ± 8 × 10
4 s
1; see
"Discussion" for rate constant nomenclature) and a second slower
phase (k3; 2.1 × 10
3 ± 2 × 10
4 s
1). ColB binding also
followed biphasic kinetics (Fig. 4A) with faster initial
(k5; 2.1 × 10
2 ± 1 × 10
3 s
1) and a slower secondary
(k7; 3 × 10
4 ± 1 × 10
4 s
1) components. Thus FeEnt and ColB
both exhibited complex binding and a similar rate of initial adsorption
to purified FepA in DM; their secondary, slower binding components
differed by about 10-fold.
We also saw slow dissociation of
ColB from purified FepA, demonstrating that the long lived complex
depends solely on the receptor and ligand; other OM components or FepA
localization in the OM were not required. When FepAE280C-Fl·ColB was
incubated with excess FeEnt over an extended period, a further decrease in fluorescence gradually occurred (Fig.
5) that followed a simple exponential
decay. It was slower (k10; 6.7 × 104 ± 4 × 10
6 s
1,
t1/2 = 17 min; Fig. 5) than the predicted off-rate,
but 10-fold faster than ColB release in the absence of FeEnt in
vivo. This decrease in fluorescence reflected the conversion of
the FepA·ColB complex to the FepA·FeEnt complex. It occurred slower
than the rate of FeEnt binding and probably approximates
koff for ColB in vitro. Likewise,
addition of excess ColB to FepAE280C-Fl·FeEnt slowly increased
fluorescence intensity (Fig. 6), again by
a first order exponential process (k9; 3.9 × 10
3 ± 6 × 10
5 s
1,
t1/2 = 3 min; Fig. 6) that reflected the conversion
of FepA·FeEnt to FepA·ColB.
Ligand Binding in Triton X-100
Ligand binding kinetics in 2% Triton X-100 were similar to those observed in DM (Table I), except that in Triton X-100 the fluorescence of FepAE280C-Fl increased approximately 15% upon ColB addition. Nevertheless, the fluorescence of FepAE280C-Fl in Triton X-100 decreased approximately 30% upon the addition of FeEnt, consistent with the results in DM, and the antagonistic behavior of the two ligands in Triton X-100 was the same as was seen in DM. The rates of fluorescence changes in Triton X-100 were very similar to those measured in DM (Table I), and in both detergents ligand binding was clearly a two-component process (see Scheme 1).
|
Affinity Purification of FepA
The avidity of FepA for its
ligands suggested affinity chromatography as a method for its
purification. ColB-Sepharose purified FepA from Triton
X-100-solubilized OM fractions of RWB18-60/pFepAE280C (Fig.
7A). It was more effective
than immunoaffinity chromatography (Fig. 7B), because 81K*
(32), the OmpT-generated degradation product of FepA, stuck tightly to
anti-FepA-Sepharose, but not to ColB-Sepharose. Second, a mild salt
gradient released FepA from ColB-Sepharose, while only high
concentrations of the chaotropic agent trichloroacetate eluted the
receptor from the immunoadsorbent, and these harsh conditions also
released antibodies from the resin that contaminated the FepA
product.
Affinity purification of siderophore receptors by binding to their antagonistic ligands, bacteriocins, was reported by Oudega et al. (21), who used cloacin DF13-Sepharose to purify an OM protein later recognized as the ferric aerobactin receptor, IutA (33). We reproduced this technique with ColB-Sepharose, permitting large scale, rapid purification of FepA or mutant FepA proteins; 20 ml of the affinity adsorbent isolated 5-10 mg of the receptor in 1-2 days.
Guterman (29, 34) discovered two mutations, exbA and
exbB, that resulted in ColB resistance and hyperexcretion of
a diffusible inhibitor of ColB killing. exbA was later
recognized as tonB (35), which creates OM transport
deficiencies resulting in ColB resistance. exbB also
produces colicin uptake defects, and the soluble factor was identified
as FeEnt. tonB and exbB mutants secrete
enterobactin because they are unable to transport iron, making them
chronically iron-deficient (36). Later experiments showed that metal
chelates (30, 37, 38) block colicin (and bacteriophage) killing by competitive binding to common surface receptors. FeEnt, ColB, and ColD,
for example, adsorb to a common site in FepA PL5 (10). We measured the
affinities and kinetics of these binding reactions in vivo
and in vitro. The siderophore bound more tightly to FepA than ColB or ColD, suggesting that its significant overall charge (3)
and aromaticity outweigh the potential for multivalent charge interactions between the colicins and the receptor. FeEnt may bind in a
pocket of the receptor surface that is optimized to accept it in a
complementary configuration. The colicins also use this site, but
apparently as part of a broader surface area that creates weaker
overall interactions. The lower capacity of FepA for ColD, relative to
that observed for ColB and FeEnt, suggests that the larger colicin may
bind as 1 ColD molecule per FepA trimer, as opposed to 1 molecule per
FepA monomer, seen for ColB and FeEnt.
In the two nonionic detergents we employed FeEnt and ColB engendered
spectroscopically distinct states in FepA upon binding, suggesting that
the siderophore and colicins induce different effects when they complex
FepA. Conformational changes in FepA were previously observed in the
presence of FeEnt, by analysis of nitroxide probes attached at E280C
(12). The close proximity of E280C to the FepA ligand binding domain
(12) raises the possibility that the large reduction in intensity
caused by the ferric siderophore results from chemical quenching
instead of conformational changes in the receptor. However, both FeEnt
and ColB exhibited biphasic binding kinetics to FepA, which strongly
suggests a conformational change in the receptor during their
adsorption. Analyses of the two-component pseudo-first order decay of
fluorescent probes attached in PL5 (Table I) revealed several possible
equilibria (Scheme 1). In DM the initial, fast binding component was
similar for both ligands (approximately 0.02 s1), and the
secondary, slow component was about 10-fold faster for FeEnt (2.1 × 10
3 s
1) than ColB (3 × 10
4 s
1). In Triton X-100 the binding of
both ligands was also biphasic, but the initial stages were slower
(approximately 0.006 s
1), and we did not observe rate
differences in the second stages of FeEnt and ColB binding (Table I).
These data suggest a detergent effect on the binding reaction (see also
below).
The mutual exclusion of one ligand by the binding of the other allowed
us to measure their dissociation rates from FepA. In contrast to the
biphasic association kinetics observed for FeEnt and ColB, their
dissociation rates in the presence of the other followed
single-component first order decays. The release of the siderophore and
the binding of the colicin, or vice versa, may follow one of two
pathways. In a microscopically reversible model, the two-stage FeEnt
binding pathway may simply revert to release the siderophore and free
FepA for ColB binding. In this case the rate constant for release of
FeEnt in the presence of ColB (k9) reflects the
reversal of the steps leading to FeEnt binding
(k4 and k2) followed by
the forward progress of ColB binding (k5 and k7). Then k9 reflects the
rate-limiting step among k4,
k2, k5, and
k7. If the ligand exchange reaction proceeds
along this sequential pathway, then the individual steps must occur as
rapidly as the overall dissociation rate (i.e.
k4, k2,
k5, and k7 must be
k9). This condition was met in Triton X-100,
but not in DM (k7 was
k9). A possible explanation for this
difference is that in DM the rapid release of FeEnt from FepA
(k9) results from an active solubilization of
the siderophore off the surface of the receptor by the detergent. For
release of ColB in the presence of FeEnt, k10
reflects the rate-limiting step among k8,
k6, k1, and
k3. In this case the individual steps occurred
as rapidly as the observed dissociation rate
(k6, k8,
k1, and k3 were
>k10), in both DM and Triton X-100. Thus, the
microscopically reversible pathway accounts for the dissociation
kinetics of both ligands in Triton X-100, and the release of ColB in
DM, and is the most likely explanation of the observed dissociation
kinetics in both detergents. An alternative mechanism for the rapid
release of FeEnt in the presence of ColB involves the binding of the
toxin to FepA·FeEnt, creating a ternary complex (FepA·FeEnt·ColB)
that dislodges the siderophore. Our data tends to exclude this
mechanism, because no ColB binding was seen in the presence of FeEnt.
Neither did we obtain evidence of a ternary complex in vivo,
in [59Fe]Ent binding experiments in the presence of
saturating ColB (data not shown). Thus, our results are most consistent
with full release of one ligand before binding of another.
One or more intermediate states must exist in the transport mechanism of a ligand-gated channel like FepA, in which the solute initially binds to an external region of the receptor. Our results show the existence of a second phase in the initial adsorption stage, that is energy- and TonB-independent, and precedes the TonB-dependent internalization of ligands. The likely explanation is that a conformational change occurs in FepA as a second step of binding, as previously suggested by site-directed spin labeling of FepA in vitro (12). Such an intermediate complex also explains the discrepancy between the Kd of FeEnt binding (20 nM) and the Km of FeEnt transport (200 NM) (10); when an initial rapid binding occurs, followed by other intermediate steps, the Km deviates from the Kd according to the rate constants for formation and collapse of the intermediates. In subsequent stages the conformation of FepA surface loops change to "open" the receptor, and the ligand enters the underlying channel (6). This internalization reaction, which may itself contain numerous distinct steps, ultimately requires energy expenditure and the participation of TonB to reach completion.
Like the siderophore, transport of the colicin occurs in two stages: binding to the cell surface, and passage through the OM bilayer. Again, our data address the energy-independent, biphasic first stage of adsorption to FepA. The steps after binding remain ill defined. FepA·ColB dissociates more slowly than FepA·FeEnt; this slow dissociating complex likely results from conformational changes in the receptor and/or the ligand that hold the proteins together. The progression of ligand binding to this tight complex did not require TonB or other OM components. Subsequent, TonB-dependent conformational changes in FepA likely trigger uptake after binding, by initiating colicin passage through the FepA channel (6).