From the Department of Chemistry & Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
Received for publication, December 14, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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The ferric siderophore transporters of the
Gram-negative bacterial outer membrane manifest a unique architecture:
Their N termini fold into a globular domain that lodges within, and
physically obstructs, a transmembrane porin Gram-negative bacteria recognize and transport ferric siderophores
(1, 2) through proteins in their outer membrane
(OM1). They secrete these
chelators in the iron-deficient environments that they encounter in the
wild and in the host. Because of its involvement in cellular processes,
iron is essential to survival, and bacteria possess efficient systems
to obtain it (3-6). However, their competitors in the microbial world
parasitize these iron uptake pathways. Bacteriocins and phages bind to
siderophore receptors as an initial step in the penetration of the cell
wall (7-9). Other microbes are not the only antagonists that target
the iron portals of bacteria: synthetic antibiotics that couple
antibacterial agents to organic iron complexes also enter through
siderophore receptors (10-13). Finally, iron acquisition is crucial to
the pathogenesis of Gram-negative bacteria, including
Salmonella, Neisseria, Yersinia,
Vibrio, and Hemophilus (14-20). In
response, higher organisms defend themselves by the sequestration of
iron in proteins like ferritin, transferrin, lactoferrin, and
conalbumin (21, 22). The competition for iron in vivo is so
fierce that one pathogenic species, Borrelia, evolved
completely novel biochemical systems that do not require the metal
(23).
The OM proteins that initiate the uptake of iron, antibiotics,
colicins, and phage function as ligand-gated porins (LGP (24-26)). They contain a transmembrane channel formed by amphiphilic Their ligand recognition and transport processes, for example, are only
vaguely defined. The OM receptors of Escherichia coli discriminate numerous ferric siderophore complexes, which, despite their similar chelation geometry (hexacoordinate) and size (600-1000 Da), are distinct in composition and charge (1, 2, 42). Each ferric
siderophore enters through a different OM iron transporter. Ferric
enterobactin (FeEnt), the native E. coli siderophore,
penetrates through FepA (43), whereas ferrichrome (Fc), a fungal
product that E. coli also utilizes, passes through FhuA
(44). Does LGP specificity derive from residues in their surface loops
or from amino acids in the N-domain, at the entrance to the membrane
channel (Fig. 1)? Experiments on a FhuA mutant devoid of its N-terminal domain (45) were relevant to this issue, as well as that of ligand
transport. In those studies the N-domain of FhuA was not necessary for
Fc, colicin, and bacteriophage uptake. The experiments we report herein
duplicated and confirmed those findings on FhuA and characterized
comparable constructions from FepA, which gave almost identical
results. Furthermore, when we genetically exchanged the N termini of
FepA and FhuA, both hybrid proteins still bound and transported their
appropriate ligands.
Bacterial Strains, Plasmids, and Media--
Bacteria harboring
plasmids (Table I) carrying the genes of
interest were cultured in LB broth or MOPS minimal media (46). It was
impractical to create single-copy chromosomal derivatives of the
numerous constructs we generated. Instead, we individually transferred
all of the genes, including wild type fepA and
fhuA, to the low-copy plasmid pHSG575, which exists in
E. coli at a level of two to three copies/cell (47).
Genetic Engineering--
We used PCR to join the FepA N
terminus (residues 1-152) to the FhuA C terminus (residues 160-723),
and the FhuA N terminus (residues 1-155) to the FepA C terminus
(residues 149-724). We first cloned the N- and C-terminal domains of
fepA and fhuA and then ligated them together. For
instance, for FepNFhu Binding and Transport--
The adsorption of
[59Fe]Ent and [59Fe]Fc was measured
with metabolically inactive KDF541 (46) expressing FepA or FhuA,
respectively. [59Fe]Ent and [59Fe]Fc were
prepared at a specific activity of ~200 cpm/pM and chromatographically purified. Binding manipulations were performed at
0 °C. A mid-log bacterial culture was chilled on ice for 1 h,
and an aliquot (containing ~5 × 107 cells) was
pipetted into a 50-ml test tube and incubated on ice. 25-ml volumes of
ice-cold MOPS minimal media, containing varying concentrations of
[59Fe]-siderophore, were poured into the tubes to achieve
rapid and thorough mixing. After 1 min the binding reactions, which
were performed in triplicate, were filtered through 0.45-µm
nitrocellulose, the filters were washed with 10 ml of 0.9% LiCl
and counted in a Packard Cobra gamma counter. The initial adsorption
reaction reached equilibrium within 5 s at physiological
temperatures and within 1 min on ice. The FepA-deficient strain KDF541
was simultaneously tested as a negative control, and any nonspecific
adsorption of [59Fe]siderophores by this strain was
subtracted from the experimental samples. Whenever necessary because of
low cpm bound, the assay samples were counted for an extended period of
time (up to 30 min) to decrease standard error. Binding data were
analyzed using the Bound versus Total equation of Grafit
4.013 (Erithacus Ltd., Middlesex, UK).
Ferric siderophore uptake was qualitatively evaluated by nutrition
tests (46), and the transport of [59Fe]Ent and
[59Fe]Fc was quantitatively measured in live bacteria
(46). For ferric siderophore acquisition (and general and specific
porin-mediated transport as well (48)), the outer membrane transport
stage constitutes the rate-limiting step of the overall uptake process (4, 6, 24, 26, 46, 49-51). Periplasmic binding proteins (51) and the
components of the inner membrane permease complexes are either
constitutively expressed or de-repressed by iron starvation. Therefore,
our transport data describe the outer membrane components of the
process, FepA, FhuA, and their mutant derivatives.
Transport manipulations were performed at 37 °C. A volume of mid-log
bacterial culture (50-100 µl containing ~5 × 107
cells) was pipetted into a 50-ml test tube and incubated in a 37 °C
water bath. Without delay, 25 ml of prewarmed MOPS minimal media,
containing glucose (0.2%), appropriate nutritional supplements, and
varying concentrations of [59Fe]siderophores, were poured
into the tube to achieve rapid and thorough mixing. The transport
reactions were quenched by the addition of a 1000-fold excess of
nonradioactive ferric siderophores, immediately filtered through
0.45-µm nitrocellulose, and the filters were washed with 10 ml of
0.9% LiCl and counted in a Packard Cobra gamma counter. Kinetic
parameters were determined from the initial rates of uptake, which were
calculated at each substrate concentration from two independent
measurements made in triplicate at 5 and 15 s: cpm bound to the
cells at 5 s were subtracted from the cpm associated with the
cells at 15 s (10-s uptakes). For some mutants with low uptake
rates (FepNFhu Bacteriocin and Bacteriophage Susceptibility--
Serial
dilutions of colicins B, D, or M, and bacteriophage T5 and SDS-PAGE and Western Immunoblots--
Proteins were
separated on SDS-PAGE gels (52), and either stained with Coomassie Blue
or electrophoretically transferred to nitrocellulose paper. Western
immunoblots were incubated with anti-FepA mAbs 26 or 45 (52), developed
with 125I-protein A (49), quantitated by image analysis
with a Packard Instant Imager, and visualized by exposure of x-ray
films. The former antibody recognizes an epitope in the N-terminal
globular domain of FepA (bounded by residues 27and 37 (52)); the latter binds in loop 4 of the C-terminal barrel domain, near residue 329 (49,
52). For native gel electrophoresis, 50 µg of OM or 2 µg of
purified protein was resuspended in LDS sample buffer and sonicated in
an ice water bath for 5 min. After a 1-min centrifugation in a
microcentrifuge, the samples were electrophoresed overnight at 5 mA and 4 °C on LDS-PAGE gels (53).
Mutant Protein Expression and Localization--
The expression
of FepA and its mutants was quantitated by Western immunoblots of whole
cell lysates with anti-FepA monoclonal antibodies 26 and 45 and
125I-protein A. Protein concentrations were determined by
image analysis, relative to standards. The localization of the chimeric
proteins was determined by fractionation of inner and outer membranes
on sucrose gradients (54), followed by Western immunoblots of the fractions.
Enzyme-linked Immunosorbent Assays (ELISA)--
Purified OM
fragments or purified, denatured FepA, were suspended in 10 mM ammonium acetate, 10 mM ammonium carbonate,
pH 8.3, at 10 and 0.5 µg/ml, respectively, dispensed into microtiter plates (Immulon), and incubated at 4 °C overnight. All further incubation steps were performed at 25 °C. In the morning, excess or
unabsorbed antigen was removed by three washes with Tris-buffered saline containing 0.05% Tween 20, pH 7.4 (TBS-Tween), and the plates
were blocked with 2% bovine serum albumin in TBS for 30 min. The
plates were washed three times with TBS-Tween, and serial dilutions of
anti-FepA mAbs in 50 µl of blocking buffer were added and incubated
for 1 h. The blocking buffer was removed and 50 µl of a 1/100
dilution of goat-anti-mouse immunoglobulin-alkaline phosphatase (Sigma
Chemical Co.) in blocking buffer was added to the plates. After
incubation for 1 h at 25 °C, the plates were washed three times
and developed with 50 µl of p-nitrophenyl phosphate (1 mg/ml, Sigma). After a 1-h incubation at 25 °C, 50 µl of 2 N NaOH was added to stop the reaction and absorbance was
measured at 405 nm with a microplate reader.
OM Protein Cross-linking--
Bacteria grown in MOPS minimal
media or sucrose gradient-purified OM fractions were suspended at
109 cells/ml or 10 mg/ml, respectively, in 4 mM
SulfoEGS, a water-soluble homobifunctional cross-linker
(Mr 661 Da) that preferentially reacts with
primary amines, for 2 h at 0 °C. The compound contains a 16-Å
spacer arm, which is cleaved by reaction with hydroxylamine. After
cross-linking, cells or OM proteins were solubilized in sample buffer,
subjected to SDS-PAGE, and stained with Coomassie Blue. When indicated,
5 µM ferric enterobactin was added to the cells prior to
cross-linking. Cross-linked bands were excised from the gels, cleaved
with hydroxylamine, electroeluted, and re-electrophoresed: The identity
of the cross-linked proteins was determined by sequence analysis of
their N-terminal 15 residues (Protein and Nucleic Acid Sequence
Facility, Medical College of Wisconsin).
Genetic Exchange of LGP N Termini--
To understand the basis of
ligand specificity in FepA and FhuA, we switched their N-terminal
domains and biochemically characterized the resulting hybrid proteins.
Although Braun et al. (45) reported the ability of an
N-terminal deletion of FhuA to transport, they did not consider the
affinity of the mutant for its ligands, nor the kinetic parameters of
the uptake process. Our experiments duplicated their construction,
created comparable deletion mutants of FepA, and created the two
chimeric proteins that switched the N-domains of FepA and FhuA. In the
latter case, the protein engineering conserved three structural
features of FepA and FhuA: the globular N-domain, the
The hybrid proteins functioned with unexpected efficiency and
selectivity, conferred by the
The constructs that deleted the N-domains of FepA and FhuA (Fep Interactions with Colicins and Bacteriophage--
The The N-domain in Transport--
The occlusion of the FepA and FhuA
channels by their own N termini raises another question: What is the
function and structural disposition of the N-domain during transport?
Passage of ferric siderophores requires a minimum diameter of about 15 Å, which does not exist in either FepA or FhuA. Does the N-terminal
domain exit the
Uptake experiments with the mutant proteins, especially those
containing the FhuA barrel, bore relevance to these questions. Despite
its binding ability, the presence of the FepA N terminus within it made
it unlikely for FepNFhu Conformation of the Hybrid Proteins--
The tertiary structures
of the mutant proteins were pertinent to the understanding of their
binding and transport abilities. We characterized the conformations of
the chimeric proteins by native electrophoresis, immunochemistry, and
nonspecific permeability. The
As a further indication of the folding of FepNFhu
Finally, neither chimeric protein conferred increased permeability to
large antibiotics, whereas bacteria expressing either Fep Cross-linking with SulfoEGS--
In the presence of formaldehyde,
FepA cross-links to another cell envelope protein, TonB (for review see
Ref. 4). However, skepticism about formaldehyde as an indicator of
proximity led us to test other cross-linking agents with more specific
chemical targets. The bifunctional, cleavable cross-linking agent
SulfoEGS selectively reacts with primary amines and is too large (661 kDa) to pass through the aqueous channels of the OM, restricting its chemical action to Lys residues on the membrane surface. When applied
to live bacteria or purified Escherichia coli outer
membranes, SulfoEGS generated two prominent protein complexes that
included FepA, of approximate molecular masses 100 (band
1) and 120 kDa (band 2; Fig.
6). The identity of the cross-linked
protein(s) in the 100-kDa complex is currently unknown. When cleaved,
the 120-kDa product yielded, in addition to FepA, the major OM proteins OmpF/C and OmpA, as determined by Edman degradations of their N-terminal 15 residues (Fig. 6). The ostensible discrepancy in the
concentration of FepA, OmpF/C and OmpA in lane 9 of Fig. 6 occurs because the molecular mass of FepA (81 kDa) is almost 3-fold higher than those of OmpF/C or OmpA. Accordingly, in SDS-PAGE of a
(dissociated) 1:1 complex of FepA·OmpA, for example, a nearly 3-fold lower intensity is expected for the OmpA band. Two conditions influenced the cross-linking reaction between FepA and the major OM
proteins. First, both in vivo and in vitro the
binding of FeEnt to FepA eliminated band 2, and drastically
reduced the level of band 1. The removal of the N-domain, in
Fep Since its discovery (39, 40), the TonB dependence of ferric
siderophore acquisition has remained obscure and controversial. The
crystal structures of FepA and FhuA did not explain this aspect of
ligand uptake, although a change in the structure of the N-terminal extremity of FhuA during Fc binding (28, 29) raised the possibility of
conformational signaling between the receptor protein and TonB, or
another component of the transport system. The localization of the
"TonB-box" of siderophore transporters within the region of the N
terminus that changed conformation in response to ligand binding was
superficially consistent with this notion of signal transduction
between the bound receptor protein and TonB. In this light it was
unexpected that a mutant FhuA protein devoid of the N-terminal globular
domain effectively transported Fc (45). Our experiments had several
objectives relevant to this phenomenon: (i) to verify the phenotype of
the N-domain deletion of FhuA, (ii) to determine whether equivalent
mutants of FepA transport FeEnt, (iii) to consider the functional
exchangeability of the FepA and FhuA N termini, and (iv) to provide
thermodynamic and kinetic descriptions of the binding and transport
reactions by such mutant receptors.
Equilibrium binding studies unequivocally demonstrated that the
specificity of LGP, and their high affinity for ferric siderophores, derive from their surface loops. The abilities of both Fep One of the mutant constructs (FhuNFep-barrel formed by
their C termini. We exchanged and deleted the N termini of two such
siderophore receptors, FepA and FhuA, which recognize and transport
ferric enterobactin and ferrichrome, respectively. The resultant
chimeric proteins and empty
-barrels avidly bound appropriate
ligands, including iron complexes, protein toxins, and viruses. Thus,
the ability to recognize and discriminate these molecules fully
originates in the transmembrane
-barrel domain. Both the hybrid and
the deletion proteins also transported the ferric siderophore that they
bound. The FepA constructs showed less transport activity than wild
type receptor protein, but the FhuA constructs functioned with turnover
numbers that were equivalent to wild type. The mutant proteins
displayed the full range of transport functionalities, despite their
aberrant or missing N termini, confirming (Braun, M., Killmann, H., and
Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the
globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a
transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the
channel concomitant with solute internalization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strands that project to the cell surface as large loops (27-29). In this sense
the proteins are fundamentally porins in nature (30-32). However, LGP
differ from other porins, because they bind the molecules they
transport with high affinity and because they contain a globular N
terminus that resides within their transmembrane channel (Fig. 1). When
ligands adsorb to siderophore receptors, they trigger unknown events
that induce their own transport into the cell (33-35). In this sense
the receptors are ligand-gated. Finally, LGP transport requires proton
motive force (36-38) and the participation of another cell envelope
protein, TonB (39-41), which presumably promotes the opening of the
closed channels so ligands may enter. The crystal structures of
the ferric enterobactin receptor, FepA (27), and the ferrichrome
receptor, FhuA (28, 29), defined the architecture of siderophore
transporters but did not answer several pressing questions about their
biochemical mechanisms.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains and plasmids
, we PCR-amplified the DNA encoding FepN,
incorporating PstI and BamHI sites in the forward
and reverse primers, respectively, and inserted the product into the
low copy plasmid pHSG575 (47). We then PCR-amplified the DNA encoding
Fhu
, flanked by BamHI and SacI restriction
sites, and ligated it to FepN DNA in pHSG575. Additionally, we employed oligonucleotide-directed mutagenesis (QuikChange, Stratagene, San
Diego, CA) to delete residues 17-150 of fepA (creating
Fep
), residues 3-150 of fepA (Fep
2), and residues
5-160 of fhuA (Fhu
). Except when noted, fepA,
fhuA, and their derivatives were all analyzed on pHSG575,
which was used for binding, nutrition, and transport experiments.
, FhuNFep
, Fep
) we extended the uptake
period to 60 min (6). In all transport experiments, the FepA-deficient
strain KDF541 was simultaneously tested as a negative control, and any
nonspecific adsorption of [59Fe]Ent by this strain was
subtracted from the experimental samples. Transport results were
analyzed according to the Michaelis-Menten equation, using Grafit
4.013.
80 were
prepared in LB broth in microtiter plates, and 5-µl volumes of the
dilutions were transferred to LB plates seeded with the strain of
interest, using a Clonemaster (Immusine Corp., San Leandro, CA). The
titer of the phage and colicins was expressed as the reciprocal of the
highest dilution that cleared the bacterial lawn.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel of the
C-domain, and the
-turn that joins them (Fig.
1). The resulting clones avoided the
deletion or introduction of amino acids in the junction sequence:
FepNFhu
contained the N terminus and
-turn of FepA connected to
the
-barrel of FhuA; FhuNFep
contained the N terminus of FhuA
linked to the
-turn and
-barrel of FepA.
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Fig. 1.
Crystal structures of FepA (top
left), FhuA (top right), and the chimeric
constructions FhuNFep (bottom
left) and FepNFhu
(bottom
right). The N-terminal domains of FepA and FhuA
are red and orange, their
-barrels are
green and blue, and the
-turns between these
regions are cyan and magenta, respectively. The
junction regions are also shown in space filling representations,
including Arg and Lys (green), Ser and Thr
(white), and Glu (cyan); Pro and Gly are
gray; Trp is purple.
-barrels and their loops. The N-terminal domains did not affect the discrimination of ferric siderophores. FhuNFep
bound FeEnt but not Fc, whereas FepNFhu
bound Fc but not FeEnt. FepA adsorbs FeEnt with subnanomolar affinity, which persevered in the FhuNFep
chimera: The Kd
of its binding reaction with FeEnt was 0.2 nM (Fig.
2, Table
II). The reverse hybrid
(FepNFhu
) bound Fc with equivalent affinity to that of wild type
FhuA: The Kd of its
binding reaction with Fc was 0.6 nM (Fig. 2, Table
II). So, despite their heterologous N
termini, the chimeras maintained the selectivity of their
-barrels, with the affinity of wild type receptors.
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Fig. 2.
FeEnt and Fc binding. The adsorption of
[59Fe]Ent (open symbols) and
[59Fe]Fc (filled symbols) was measured with
metabolically inactive KDF541 expressing FepA or FhuA ( ), FhuNFep
(
), FepNFhu
(
), Fep
(
), and Fhu
(
), carried on
pHSG575.
Biochemical properties of FepA and FepA mutants
and
Fhu
) also bound ferric siderophores with wild type affinities (Fig.
2 and Table II). Our Fhu
construct was identical in composition to
that of Braun et al. (45). Regarding the
-barrel-only
clones of FepA, we engineered two different derivatives. In
fepA
17-151 (Fep
) we removed most of the the
N-domain but left amino acids 1-16, which include the TonB-box region,
fused to Gly-151. Besides Fep
we engineered
fepA
3-151 (Fep
2), which eliminated the TonB box. Except for a slight difference in expression (Fep
was better), the two clones functioned identically in binding, nutrition, and transport assays; we only report data for Fep
. The empty
-barrels of FhuA and FepA avidly bound their appropriate ferric siderophore, indicating that the surface loops select the iron complexes that the
bacteria encounter in their environments: the loops identify correct
iron chelates and reject improper ones. Although they were irrelevant
to ferric siderophore recognition, the N-terminal domains of FepA and
FhuA were needed for optimum binding. Bacteria-expressing constructions
that exchanged or deleted them adsorbed ferric siderophores with much
lower capacities (Fig. 2 and Table II). This reduction did not result
from poor expression (Table II), improper localization, or instability
(Fig. 4): rather, biochemical characterizations indicated that only a
fraction (~10%) of the mutant proteins bound ferric siderophores.
Thus, a correct N-domain, which was not needed for discrimination of
the metal chelates, was essential for their maximal binding. Without
their homologous N-domains, mutants of FepA and FhuA adsorbed much
lower amounts of ferric siderophores to the cells.
-barrels
of the mutant proteins also dictated interactions with bacteriocins and
bacteriophages. The FepA
-barrel conferred susceptibility to
colicins B and D, but not M or the phages that use FhuA, again
demonstrating the irrelevance of the N-domain to ligand selection.
Likewise, the FhuA barrel recognized T5 and
80, and the
transposition of the FepA N terminus into it did not change this
specificity (Table II). So, the antagonism between iron chelates and
noxious agents for adsorption to FepA and FhuA derives from competition
for structural elements in their
-barrels and loops, in accord with
the prior localization of residues in FepA that are needed for
efficient colicin reception. Yet, the exchange of N termini reduced the
efficiency of colicin B and D killing, suggesting that these
bacteriocins further interact with the globular domain during the
implementation of their toxicity. Unlike Braun et al. (45),
we could not detect the ability of Fhu
to confer susceptibility to
colicin M. This discrepancy may stem from the low activity of our
colicin M preparation.
-barrel during metal uptake, or change shape within the pore to create a sufficient opening for ligand transit? The former
mechanism faces an energetic barrier, because the crystal structures of
FepA and FhuA describe many residues in appropriate positions for
hydrogen bonds between the N-domains and barrel walls. The latter
notion, that the conformation of the N terminus temporarily changes to
create a pore, appears more plausible, but transient channels of such
magnitude have not been observed in membrane proteins. Furthermore,
although colicins generally comprise a slender, elongated shape, their
entry into or passage through even an open porin channel faces severe
steric obstacles (55).
to transport Fc. Yet the chimera efficiently
internalized the hydroxamate siderophore. Fc stimulated the growth of
bacteria expressing FepNFhu
as much as strains producing FhuA (Fig.
3A), in a
TonB-dependent manner. Quantitative determinations
explained this result: despite a slightly lower overall uptake affinity
for Fc than FhuA (Km values of 2.5 and 0.6 nM, respectively; Fig. 2 and Table II), the chimeric protein displayed a comparable turnover number to the wild type protein
(k3 values of 6.5/min and 4/min, respectively;
Table II). Furthermore, the isolated FhuA
-barrel transported Fc
with similar affinity (Km = 3.6 nM) and
at a faster rate (k3 = 8.3/min). Thus neither
was an N-domain required for Fc uptake, nor did the introduction of the
FepA N terminus into the FhuA barrel impair the internalization
reaction: the 160 amino acids of the globular domain were superfluous
to Fc transport. Mutant proteins containing the FepA
-barrel were
less efficient, but they corroborated the dispensability of the N
terminus to transport: Both FhuNFep
and Fep
conferred
TonB-dependent FeEnt uptake, albeit slower than wild type
FepA (k3 values of 0.9/min, 0.3/min, and
6.4/min, respectively; Fig. 3 and Table II). The low transport rates of
FhuNFep
and Fep
explained their marginal abilities in nutrition
tests (Fig. 3a). All of the plasmids failed to support
growth in KDF571 ((24) tonB; Fig. 3). We also constructed
and analyzed a second, similar chimera, FhuNFep
2, containing the N
terminus and turn of FhuA linked to the barrel of FepA, whose
biochemical properties were equivalent to those of FhuNFep
(data not
shown). Accuracy was not compromised in the binding and transport
measurements of strains with low capacity (FepNFhu
, FhuNFep
,
Fep
, and Fhu
). The 59Fe binding and uptake assays
were sufficiently sensitive to quantitate even slight binding relative
to the negative controls, which are absolute (46, 49). The close
conformity of the data to nonlinear fit calculations from the Bound
versus Total and Michaelis-Menten equations (Figs. 2 and 3,
insets) substantiated the accuracy of the measurements. For
bacteria expressing FepNFhu
, FhuNFep
, and Fep
, the samples
with the lowest capacities, the mean standard error of affinity
measurements (Kd and Km) was 9.5%. For the same strains the mean standard error of capacity and
Vmax measurements was 2.3%.
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Fig. 3.
FeEnt and Fc transport. Top,
ferric siderophore uptake was qualitatively evaluated by nutrition
tests. All of the plasmids failed to support growth in the
tonB strain KDF571, producing tests identical to that of
KDF541. Bottom, the transport of [59Fe]Ent
(open symbols) and [59Fe]Fc (filled
symbols) was quantitated in live bacteria. Strains and their
symbols are the same as in Fig. 2, except (inset) uptake by
KDF571 (tonB) harboring pFep (x) and
pFhuNFep
(asterisks).
-barrels of OM proteins are resistant
to denaturation by SDS, and they have a compact native structure that
imparts enhanced mobility in PAGE (56, 57). Denaturation of OM proteins
by heating in ionic detergents eliminates this globular structure, reverting the abnormal electrophoretic behavior. In their native states
FepA and FhuA exhibit the same compact shape and increased electrophoretic migration (52, 58), but they are less stable than
general or specific porins (59), and therefore more sensitive to
denaturation. For these reasons, electrophoretic behavior was a
revealing measure of the tertiary structure of the chimeric proteins.
When electrophoresed in LDS at 4 °C, FhuNFep
and FepNFhu
displayed the same rapid mobility as FepA and FhuA, respectively (Fig.
4), intimating that all four proteins
possessed a similarly compact globular structure.
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Fig. 4.
Expression, localization, and nondenaturing
electrophoresis of mutant proteins. A, expression.
Western immunoblots stained with anti-FepA mAbs 26 (top) and
45 (bottom) and developed with 125I-protein A. Lanes 1-3, 1, 3, and 5 µg of purified FepA, respectively;
lanes 4-10 each contain lysates of 2. 5 × 108 bacterial cells, except lane 9, which
contained 108 cells. Lane 4, KDF541
(fepA, fhuA); 5, KDF541/pFepNFhu ;
6, KDF541/pFhuNFep
; 7, KDF541/pITS23
(fepA+); 8, BN1071
(fepA+, fhuA+);
9, KDF541/pITS449 (fepA+);
10, KDF541/pFep
. Expression levels were quantitated by
image analysis and tabulated in Table II. B, localization.
Inner and outer membranes from bacteria expressing the chimeric
proteins were fractionated on sucrose gradients, and 30 µg of each
sample was analyzed by Western immunoblots with monoclonal antibodies
26 (top) and 45 (bottom). Lane 1,
KDF541 cell lysate; lanes 2-4, KDF541/pITS23 lysate, IM and
OM, respectively; lanes 5-7, KDF541/pFhuNFep
lysate, IM
and OM; lanes 8-10, KDF541/pFepNFhu
lysate, IM and OM.
C, Nondenaturing LDS-PAGE. The left panel is a
Coomassie Blue-stained, nondenaturing LDS-PAGE gel; the right
panel is a Western immunoblot of a nondenaturing LDS-PAGE gel,
stained with a mixture of anti-FepA mAbs 26 and 45 and developed with
125I-protein A. Lane 1, molecular mass markers
(Bio-Rad, Hercules CA); lanes 2 and 3, purified,
denatured FepA and FhuA, respectively; lanes 4 and
5, purified, nondenatured FepA and FhuA, respectively;
lanes 6-8, OM fragments (30 µg) from KDF541 expressing
FepA, FhuNFep
, and FepNFhu
.
, we determined the
accessibility of epitopes within its (FepA) N terminus to mAb binding.
In ELISAs utilizing four different anti-FepA mAbs that bind epitopes in
the N-terminal 150 residues, FepNFhu
was indistinguishable from wild
type FepA (Fig. 5). The antibodies against the N-domain did not react with either protein unless the
antigen was denatured before adsorption to the plates, suggesting that
the N-domain of the chimeric protein was sequestered within Fhu
, in
a comparable manner to the N-domain within the barrel of the wild type
protein. The unavailability of antibodies to the FhuA N
terminus2 precluded a similar
analysis of FhuNFep
. However, we cytofluorimetrically assessed its
proper folding, with anti-FepA mAbs against surface epitopes in the
loops of its (FepA) C-terminal domain. Like FepNFhu
in ELISA,
FhuNFep
was indistinguishable from wild type FepA in flow cytometry
(Table II).
View larger version (14K):
[in a new window]
Fig. 5.
Analysis of FepNFhu
by ELISA. Outer membranes from KDF541 carrying pITS23
(A) or pFepNFhu
(B), or purified, denatured
FepA (C) were adsorbed to microtiter plates and tested with
anti-FepA mAbs 1 (
), 12 (
), 27 (
), 41 (
), 33 (
), and 45 (
), or normal mouse serum (x). The first four antibodies
recognize different epitopes in the N-terminal globular domain, whereas
the latter two bind epitopes in surface loops of the barrel.
A and B plot mean values from three experiments;
C shows the results of a single experiment.
or Fhu
showed enhanced susceptibility to bacitracin (1500 Da; Table II), as
expected for OM proteins containing a large, open channel. In total,
the three different approaches all suggested that both hybrid proteins
assembled with N termini lodged within their heterologous
-barrels.
, produced analogous results, reducing the levels of the 100- and
120-kDa products to barely detectable levels (Fig. 6).
View larger version (59K):
[in a new window]
Fig. 6.
Cross-linking of bacteria with SulfoEGS.
A, outer membranes from BN1071 (lanes 1-3) or
KDF541/pITS449 (lanes 4-6) were incubated with the
cleavable cross-linker Sulfo-EGS, subjected to SDS-PAGE, and stained
with Coomassie Blue. Lanes 1 and 4, no
cross-linking; lanes 2 and 5, cross-linked by
SulfoEGS. Lanes 3 and 6, cross-linked by SulfoEGS
in the presence of 5 µM FeEnt. Lane 7,
molecular mass standards myosin (200 kDa), -galactosidase (116 kDa),
phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa). Lane 8, purified
FepA. Lane 9, band 2 in lane 2 was
excised, cleaved with hydroxylamine, electroeluted, and
re-electrophoresed: Note the resulting proteins that migrate with
apparent molecular masses of 34 and 36 kDa, whose identity as OmpF/C
and OmpA was verified by sequence analysis of their N-terminal 15 residues (data not shown). B, outer membranes from
KDF41/pITS23 (lanes 1-3), or live cells of KDF541 harboring
pITS23 (lanes 4-6) or pFep
(lanes 7-9) were
either untreated (lanes 1, 4, 7), or
cross-linked with Sulfo-EGS in the absence (lanes 2,
5, 8) or presence of 5 µM FeEnt
(lanes 3, 6, 9), and cell lysates were
transferred to nitrocellulose and analyzed by immunoblot with anti-FepA
mAb 45, developed with 125I-protein A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Fhu
to correctly discriminate FeEnt and Fc, and to bind them with
wild-type affinity, confirms this point. These data also concur with
the identification of aromatic residues in the exterior-most portion of
the loops around the entrance to the FepA vestibule, that function in
FeEnt recognition (49). In the crystal structure of FhuA, Fc lodged
deep within the vestibule interior, in contact with residues at the top
of the globular domain, suggesting that the N-domain contributes to the
overall efficacy and specificity of the ligand binding process.
However, the subnanomolar binding affinity of the two proteins that
lack the N-domain, Fhu
(for Fc) and Fep
(for FeEnt), indicate
that in both receptor proteins the globular domain does not
significantly affect ligand recognition or binding. The correct
adsorption of colicins and phages by both chimeras reiterates that, if
the N-domain acts at all in the binding reaction, then it's
contributions are minimal and secondary to those of the surface loops.
) had a lower expression level,
and others conferred lower binding capacities than wild type FepA. To
compare the uptake rates of the strains, it was therefore necessary to
determine the number of molecules of ligand transported per receptor
protein per unit of time. k3 describes this
rate, in essence the turnover number of each of the transport proteins.
It derives from the simple Michaelis-Menten description of the
siderophore uptake reaction as follows.
k3 is the only measure available by which
to compare, in vivo, populations of proteins with different
capacities and concentrations in the membrane. In our study
k3 is exactly equivalent to the term
kcat, the recommended basis for rate comparisons
of enzymes: k3 = Vmax/Cap; kcat = Vmax/ET.
(Eq. 1)
Besides its high affinity, Fhu transported Fc in a
TonB-dependent manner, with a better turnover number than
wild type FhuA (k3 in Table II). Although the
comparable FepA derivatives had reduced transport rates, their
activities must be related to the following series of controls:
fepA bacteria, tonB bacteria, and fepA+, energy-depleted bacteria. These control
strains/conditions show absolute inability to transport any of the
relevant ligands (Table II and Fig. 3 (see also Refs. 24, 46, 49, 50)).
Therefore, even the least active construct, FhuNFep
, showed the
complete range of functionalities. These data lead to the same
conclusion as that reached by others (45): The N termini of FhuA and
FepA are not needed for the transport of Fc and FeEnt. Besides their importance to the understanding of ferric siderophore transport (see
below), these data demonstrate that TonB does not function by an
interaction with the N-terminal 150 amino acids of ferric siderophore
receptors, despite indirect evidence from genetic suppression (60-62),
cross-linking (63-68), and affinity chromatography (69). Thus the
concepts of transmembrane signaling and/or functional interactions
between LGP and TonB, through the TonB-box, are specious. Instead, the
results suggest the contrary, that, subsequent to the binding of metal
chelates, the surface loops of LGP undergo TonB-dependent
conformational changes during the transport of metal chelates. This
conclusion from genetic engineering concurs with biophysical studies of
loop conformational dynamics in vivo (25).
Also contrary to previous suppositions (27-29, 70) the globular domain
apparently does not create a siderophore-specific transit pathway to
the periplasm. The idea of a substrate-customized channel conflicts
with the efficient internalization of the neutral Fc iron complex by
FepNFhu, which contains an N-domain adapted to a negatively charged,
catecholate ferric siderophore. The heterologous N-domain of FepNFhu
did not impair Fc uptake: like Fhu
, the chimera transported at a
faster rate than wild type FhuA. The FeEnt transport activity of
FhuNFep
reiterated this point. The ability of the chimeras to
internalize the ferric siderophore that their
-barrel domains
adsorbed undercuts the notion of a transient, ligand-specific pathway
to the periplasm. On the contrary, bound metal chelates enter through a
nonspecific route, neither created nor regulated by the N terminus.
If it does not act in solute recognition or uptake, then what is the
function of the N terminus? Constructs without an N-domain, or with an
aberrant one, adsorbed ferric siderophores to much lower capacities
than the wild type receptors, suggesting that in the native proteins
the proper insertion of the N-domain into the barrel facilitates
binding. Such an effect may occur by protein·protein interactions
between the globular domain and the barrel, which optimize the
conformation of surface loops. In the populations of empty -barrel
proteins that we studied only a fraction (~10%) of the molecules
bound ferric siderophores. However, the percentage of receptor proteins
that adopted an appropriate conformation, perhaps by random
conformational motion (the flexibility of the loops of FepA was
apparent in its x-ray structure determination), attained wild type
affinity. So, despite the fact that it does not act in ligand
discrimination, the globular domain enhances the binding capacity of
ferric siderophore receptors. In this sense though, the N-domains of
FepA and FhuA were not interchangeable, because like Fep
and Fhu
the chimeric receptors also showed reduced binding capacity. We
addressed the possibility that in FepNFhu
and FhuNFep
the
globular domains do not enter the porin
-barrels but remain
suspended in the periplasm. Electrophoretic and immunochemical
characterizations argued against this alternative view of their
structure: Both hybrid proteins were identical to wild type FepA and
FhuA in these tests. It is therefore likely that the globular domains
of the chimeras reside within their
-barrels, but the absence of
appropriate interactions between the heterologous N- and C-domains
prevents the attainment of wild type binding capacity.
In addition to its function in the optimization of ferric siderophore
binding, the presence of the globular domain within the -barrel may
preserve the integrity of the cell envelope to noxious small molecules.
The bacterial OM creates a selective permeability barrier that permits
uptake of nutrients and vitamins, generally smaller in mass than 600 Da, and excludes larger, toxic, hydrophobic molecules, including the
natural detergents of the animal gut (48). In this light the
obstruction of the large LGP channel, which when open renders the
bacteria susceptible to bile salts in the gut, is an important
rationale for the arrangement of the binding-receptive state that we
propose. This general trend of OM protein design appears in all other
structurally characterized porins, including the E. coli
transporters OmpF and LamB, which both contain a uniquely oriented loop
(L3) that restricts permeability through their channels (30-32).
The crystal structure of FhuA created controversy about potential
conformational changes that may occur in the surface loops of LGP
during ferric siderophore binding and transport. Although ample
evidence from biophysical studies suggested structural dynamics within
FepA (25, 33, 71, 72) and FhuA (34) the absence of different loop
conformations in the crystals of ligand-bound and ligand-free FhuA
challenged this deduction. Nevertheless, it is most relevant to
consider conformational change in the in vivo environment,
free from the constraints of crystallography. A crystal structure
captures only one of the many conformational states that a protein may
adopt, as aptly stated by Gutfreund: " ... from a picture of a
racehorse, you can't tell how fast it can run" (73). The
cross-linking experiments reported herein provide further
contradictions to the static picture surmised from crystallography.
When live bacteria were exposed to sulfoEGS, FepA cross-linked to the
major OM proteins OmpF/C and OmpA. Binding of FeEnt eliminated the
cross-linking, as did the removal of the N-domain, in Fep. The
experiment demonstrates two loop conformations in vivo: an
unoccupied form in which the surface loops reach close proximity to
contiguous OM proteins, and a bound form with closed loops that do not
cross-link to other proteins. The failure of Fep
to cross-link to
OmpF/C and OmpA suggests the need for the N-domain to enter the barrel
to create the open state.
We've previously objected to the use of formaldehyde cross-linking in the study of proposed physical interactions between TonB and FepA, because of a lack of control data on the cross-linking of TonB to non-TonB-dependent OM proteins, uncertainties about the nature and identities of chemical targets of the formaldehyde-activated residues within the cross-linked proteins, and the absence of quantitation of the very small amount of total cross-linked products (4). The cross-linking experiments we report attempted to satisfy these same concerns, by employing a reagent with well defined targets and methods (Coomassie Blue-stained SDS-PAGE gels and 125I-protein-A immunoblots) that were amenable to the identification of all detectable cross-linked products and their precise quantitation. In our experiments up to 70% of the total FepA in the OM became involved in cross-links. Band 2 (Fig. 6) was abundant enough that we excised it from gels and determined the N-terminal sequences of its components.
The several experimentally defined forms of LGP provide insight
into the iron transport process. First, structural differences exist
between the unbound and bound receptor protein, implicating conformational change as a main element of the ligand binding process.
During ferric siderophore acquisition LGP likely progress between
multiple conformations; at least one of the transitions between
structural forms requires energy and/or the participation of TonB.
According to this view, FepA initially assumes an "open" state,
with the N-domain inside the barrel, where it compels the unoccupied
loops to an extended, optimized configuration for binding. FeEnt
adsorption to the loops stimulates their coalescence, creating a
"closed" complex. Besides our cross-linking data, ESR
spectroscopy (33, 72) described these open and closed forms, and
kinetic fluorescence analyzes (71) showed the movement of FeEnt between two distinct sites during binding. Consistent with this view, site-directed mutagenesis defined residues in an exterior binding site
(Tyr-272, Phe-329) and an interior site (Arg-316 (50) and Tyr-260 (49)). After the ferric siderophore attains binding equilibrium, it sits poised above the globular N-domain, itself situated within the channel. Crystallographic experiments presumably captured and described such a closed complex of FhuA. The
transport-competent, empty -barrels of FhuA and FepA constitute
another noteworthy form of LGP. Their very existence and functionality
implies that the N-domain exits the barrel as ligands traverse the OM.
On this basis we propose that either directly or indirectly, TonB
promotes loop dynamics that force bound ligands into the underlying
pore. But this action of TonB does not occur by its binding to the
"TonB-box" or another site within the N-domain of siderophore
receptors, because neither Fhu
nor Fep
contain these regions.
Rather, we suggest that, in the native receptors the
TonB-dependent dislodgement of the bound solute, driven by
an inward contraction of the surface loops, concomitantly ejects the
N-domain and releases iron into the now open channel to the periplasm.
Such proposed movement of the N terminus in and out of the channel
superficially reprises one of the first postulates of membrane protein
function, the "ball and chain" hypothesis (74). The possibility of
hydrogen bonds between the residues on the surfaces of the globular
domain and the
-barrel walls suggests an energetic barrier to this
reaction mechanism, perhaps explaining the energy dependence of iron
transport across the OM. For FhuA and FepA such movement of the
globular domain does not cause or regulate ligand uptake but rather
occurs in response to ligand internalization. This model also
rationalizes the problematical uptake of siderophore antibiotics, which
often double the mass of an iron complex without preventing its
recognition and transport. A large, nonspecific uptake pathway,
comparable to those that exist in the open
-barrels of FepA and
FhuA, is necessary to accommodate the passage of such Trojan horse
antibiotics through the OM.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ines Chen, Paul Cook, Charles Earhart, Giovanna Ferro-Luzzi Ames, and Emil Gotschlich for reading the manuscript, T. Hashimoto-Gotoh for pHSG575, and Marjorie Montague for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB9709418 and National Institutes of Health Grant GM53836.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.
To whom correspondence should be addressed: Tel.: 405-325-4969;
Fax: 405-325-6111; E-mail: peklebba@ou.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M011282200
2 J. A. Coulton, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: OM, outer membrane; LGP, ligand-gated porin; FeEnt, ferric enterobactin; Fc, ferrichrome; mAb, monoclonal antibody; LDS, lithium dodecyl sulfate; MOPS, 4-morpholinepropanesulfonic acid; PCR, polymerase chain reaction; TBS, tris-buffered saline; SulfoEGS, ethylene glycobis(sulfosuccimidylsuccinate); ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; ESR, electron spin resonance.
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