JOURNAL OF BACTERIOLOGY, May 2001, p. 2755–2764 Vol. 183, No.

9
0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.9.2755–2764.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Characterization of In Vitro Interactions between a Truncated TonB

Protein from Escherichia coli and the Outer Membrane
Receptors FhuA and FepA
GREGORY S. MOECK† AND LUCIENNE LETELLIER*
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR CNRS 8619,
Université de Paris-Sud, F-91405, Orsay cedex, France

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Received 18 September 2000/Accepted 2 February 2001

High-affinity iron uptake in gram-negative bacteria depends upon TonB, a protein which couples the proton
motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane. To advance studies
on TonB structure and function, we expressed a recombinant form of Escherichia coli TonB lacking the
N-terminal cytoplasmic membrane anchor. This protein (H6-ⴕTonB; Mr, 24,880) was isolated in a soluble
fraction of lysed cells and was purified by virtue of a hexahistidine tag located at its N terminus. Sedimentation
experiments indicated that the H6-ⴕTonB preparation was almost monodisperse and the protein was essentially
monomeric. The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated by
size exclusion chromatography. The frictional ratio (2.0) suggested that H6-ⴕTonB adopts a highly asymmet-
rical form with an axial ratio of 15. H6-ⴕTonB captured both the ferrichrome-iron receptor FhuA and the ferric
enterobactin receptor FepA from detergent-solubilized outer membranes in vitro. Capture was enhanced by
preincubation of the receptors with their cognate ligands. Cross-linking assays with the purified proteins in
vitro demonstrated that there was preferential interaction between TonB and ligand-loaded FhuA. Purified
H6-ⴕTonB was found to be stable and thus shows promise for high-resolution structural studies.

Bacteria seeking to colonize aerobic environments at phys- terminal 160-amino-acid cork or plug that is organized into a
iological pH are faced with the practical insolubility of an globular domain and is held in the 22-strand ␤-barrel by an
essential nutrient, iron. Further limiting the availability of iron extensive network of hydrogen bonds and salt bridges. An
in vertebrate hosts are iron-chelating proteins, such as lacto- elegant structural comparison of FhuA with and without
ferrin and transferrin. Gram-negative bacteria respond to iron bound ferrichrome-iron revealed uncoiling of the switch helix
deficiency by derepressing expression of a series of high-affinity (residues 24 to 29) from the cork and striking displacement of
iron transport systems (7, 8, 52). Such systems bind iron che- certain residues (for example, Glu19 by more than 1.7 nm)
lates (siderophores and host iron-binding compounds) by upon ligand binding (17, 37). What is the outcome of trans-
means of receptors located in the outer membrane. A parallel duction of this signal across the N-terminal cork domain? An
system for uptake of vitamin B12 is induced when cobalamins earlier study identified conformational changes in an N-termi-
are scarce in the environment. Bound ligands are then trans- nal periplasmic region of FhuA in response to binding of fer-
ported into the periplasm for internalization by ATP-binding ricrocin, a ferrichrome analogue (42). Such ligand-dependent
cassette transporters located in the cytoplasmic membrane. alterations, which may be closely related to those defined by
The outer membrane transport step depends upon the elec- X-ray crystallography, are likely to promote the association
trochemical proton gradient across the cytoplasmic membrane between TonB and TonB-dependent receptors, as has been
(5, 20, 49) and upon the cytoplasmic membrane-anchored pro- demonstrated for FhuA (43) and for FepA (32), and thereby
teins TonB, ExbB, and ExbD to transduce this energy across promote ligand translocation through the receptor. Such a
the periplasm (23, 32, 35, 40, 45). To date, more than 20 outer mechanism ensures efficient expenditure of energy since TonB
membrane proteins whose functions depend upon TonB have interacts preferentially with those receptors to which ligand
been identified (57). has bound (24, 40). However, the molecular mechanism of
Recently, significant advances in understanding high-affinity energy transduction remains unclear. Kadner and colleagues
iron uptake in bacteria came from determinations of the three- (12, 13) recently identified interacting pairs of residues in the
dimensional crystal structures at atomic resolution of the fer- vitamin B12 receptor BtuB and TonB; certain cysteine substi-
richrome-iron receptor FhuA (17, 37) and the ferric-enter- tution mutations toward the N terminus of BtuB (the TonB
obactin receptor FepA (11) of Escherichia coli. Perhaps the box) were effectively disulfide cross-linked in vivo to pheno-
most surprising part of the receptor structures was the N- typically compensatory cysteine substitutions around amino
acid 160 of TonB. A recent spectroscopy study (39) monitored
the dynamics of spin-labeled BtuB variants within isolated
* Corresponding author. Mailing address: Institut de Biochimie et outer membranes. Addition of substrate resulted in rapid un-
Biophysique Moléculaire et Cellulaire, UMR CNRS 8619, Université folding of the N-terminal part of the protein in the vicinity of
de Paris-Sud, Bât. 430, F-91405, Orsay cedex, France. Phone: 33 1 6915
6429. Fax: 33 1 6985 3715. E-mail: lucienne.letellier@biomemb.u-psud
the spin labels. Combined, the results that have been obtained
.fr. demonstrate that the TonB box of BtuB cycles between con-
† Present address: PhageTech, Montreal, PQ, H2W 2N9, Canada. strained and accessible conformations and that the latter may

2755
2756 MOECK AND LETELLIER J. BACTERIOL.

allow TonB to bind to the TonB box of BtuB (13, 39). Com- (Complete) tablets and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride
plete removal of the TonB box, however, does not eliminate all (Pefabloc SC) were purchased from Roche Diagnostics (Meylan, France) and
were used as recommended by the manufacturer.
TonB-dependent receptor activity. Indeed, a FhuA variant car- Cloning of TonB. The tonB gene (46) was amplified by PCR from 20 ng of
rying a deletion of most of the cork domain (FhuA⌬5-160) and chromosomal DNA of E. coli XL-1 Blue. The reaction mixture included 1.5 U of
thus devoid of its TonB box still transported ferrichrome in a the proofreading DNA polymerase Pwo and 50 pmol (each) of primers that were
TonB- and energy-dependent manner, albeit at a rate that was designed to incorporate 5⬘ NdeI and 3⬘ XhoI sites for cloning the amplified
product into pET-28. The sequences of the PCR primers were as follows: for-
significantly lower than the wild-type FhuA rate (6).
ward primer, 5⬘-TCAGTCCATATGCATCAGGTTATTGAACTA (the NdeI
TonB is essentially a periplasmic protein with an uncleaved site is underlined; the CAT codon in bold face type encodes His33 of the TonB
hydrophobic N terminus (47) anchored in the cytoplasmic sequence [46]); and reverse primer, 5⬘-TCGATCCTCGAGTTACTGAATTTC
membrane both in Salmonella enterica serovar Typhimurium GGTGGT (the XhoI site is underlined; the TTA in boldface type is reverse and
(21) and in E. coli (50). How might TonB maintain its connec- complementary to the stop codon of the chromosomal tonB sequence and is

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preceded by the codon for the C-terminal Gln239 of the TonB sequence). PCR
tion to the cytoplasmic membrane and at the same time mod- products were trimmed with NdeI and XhoI and ligated to pET-28 that had been
ulate the activity of receptors in the outer membrane? The restricted with the same enzymes.
proline-rich regions between residues 70 and 102 of TonB from Expression and purification of soluble histidine-tagged TonB. The plasmid
E. coli ([EP]4X13[KP]6) (46) and between residues 66 and 106 of encoding H6-⬘TonB was transformed into E. coli ER2566, and 500-ml cultures in
TonB from S. enterica serovar Typhimurium ([EP]5X13[KP]7) Luria broth plus kanamycin (30 ␮g/ml) were induced with 0.5 mM isopropyl
␤-D-thiogalactopyranoside (IPTG). Cells were collected by centrifugation and
(21) adopt an extended structure (16, 21, 58) that may contrib- suspended in 50 ml of 100 mM sodium phosphate, pH 7.9. Protease inhibitor
ute to the interaction between the C-terminal 60 residues of cocktail tablets and phenylmethylsulfonyl fluoride were added, and all steps were
TonB (2, 31, 35) and outer membrane-localized receptors un- carried out at 4°C or on ice. Cells were lysed by sonication, and the cleared
der certain osmotic conditions (28). supernatant was mixed with 5 ml of equilibrated Ni2⫹-NTA agarose resin in
batch for 30 min. Protein was eluted with an imidazole step gradient and was
The N-terminal anchor also plays a critical role in the func-
dialyzed extensively against 100 mM sodium phosphate, pH 7.9.
tion of TonB (22, 25). Although certain TonB variants with Anti-TonB antibodies. White Leghorn hens (Genaxis, Montigny le Breton-
altered N termini retain the ability to bind to outer membrane neux, France) were immunized subcutaneously at 3-week intervals with 100 ␮g of
receptors (22, 29, 35), point mutations affecting this predicted purified H6-⬘TonB in Freund’s complete adjuvant. Immune serum drawn 2 weeks
␣-helical transmembrane domain disrupt interaction with the after the first boost revealed anti-TonB titers of 1:15,000 relative to the preim-
mune control serum in an enzyme-linked immunosorbent assay (ELISA) with
cytoplasmic membrane protein ExbB and abolish ferric sid- purified H6-⬘TonB. The specificity of the anti-TonB immune serum was con-
erophore transport and killing by TonB-dependent phages and firmed by blotting against whole-cell extracts of E. coli KP1060 (TonB⫹) and
protein antibiotics termed colicins (29, 32). Mutations in ExbB KP1120 (tonB).
or ExbD or overexpression of TonB without concomitant over- Characterization of H6-ⴕTonB and of the H6-ⴕTonB–FhuA.H6 complex by size
exclusion chromatography. Proteins were applied to a Superose 12 HR 30/10
expression of ExbB or ExbD increased the rate of degradation
column (Pharmacia) equilibrated either in 100 mM sodium phosphate buffer (pH
of TonB and reduced TonB-dependent receptor activity (1, 18, 7.9) or in 20 mM Tris-HCl (pH 8.0)–150 mM NaCl–0.1% lauryldimethylamine
53). Such susceptibility to endogenous proteolysis has impeded oxide (TLN buffer). The flow rate was kept at 0.25 ml/min. To calibrate the
isolation of TonB from E. coli. To overcome these difficulties, Superose 12 column, we obtained experimental values for the elution volumes
we expressed a soluble derivative of E. coli TonB lacking the (Ve) of the following standard globular proteins with known Stokes radii (Rs):
thyroglobulin (Rs ⫽ 8.6 nm; Ve ⫽ 8.8 ml), ferritin (Rs ⫽ 6.3 nm; Ve ⫽ 10.7 ml),
N-terminal cytoplasmic membrane anchor. Here we describe catalase (Rs ⫽ 5.2 nm; Ve ⫽ 11.7 ml), aldolase (Rs ⫽ 4.6 nm; Ve ⫽ 12.0 ml),
purification of this hexahistidine-tagged TonB variant (H6- bovine serum albumin (Rs ⫽ 3.5 nm; Ve ⫽ 12.5 ml), ovalbumin (Rs ⫽ 2.8 nm;
⬘TonB), show that it retains affinity for the TonB-dependent Ve ⫽ 13.4 ml), chymotrypsinogen A (Rs ⫽ 2.1 nm; Ve ⫽ 14.9 ml), and RNase A
receptors FhuA and FepA, and demonstrate that it responds to (Rs ⫽ 1.75 nm; Ve ⫽ 15.5 ml). The partition coefficient (KD) was calculated with
the equation KD ⫽ (Ve ⫺ V0)/(Vt ⫺ V0), where V0 is the void volume of the
the ligand occupation status of these receptors. The stability of
column (7.3 ml) and Vt is the total volume of the column (21.0 ml). Dextran blue
H6-⬘TonB and its ability to interact with TonB-dependent re- 2000 was used as a marker of the void volume. The total volume was measured
ceptors imply that the protein is amenable to further structure- by using NaNO3 (80 mM) (3, 34). The calibration curve Rs as a function of KD
function studies. (data not shown) was the fit obtained with a polynomial of degree 3 (correlation
coefficient ⫽ 0.989).
For size exclusion chromatography of the FhuA–H6-⬘TonB complex, hexahis-
MATERIALS AND METHODS
tidine-tagged FhuA protein (FhuA.H6) (42) was purified from TLN buffer-
Bacterial strains and reagents. E. coli XL-1 Blue [recA1 endA1 gyrA96 thi solubilized outer membrane preparations of E. coli HO830fhuA(pHX405) as
hsdR17(rK⫺ mK⫹) supE44 relA1 lac (F⬘ proAB⫹ lacIqZ⌬M15::Tn10); Stratagene, described previously (27). For experiments with ferricrocin-iron, the iron-loaded,
La Jolla, Calif.], DH5␣ [␾80dlacZ⌬M15 recA1 endA1 gyrA96 thi-1 hsdR17 (rK⫺ FhuA-specific siderophore was preincubated with samples at a twofold molar
mK⫹) phoA ␭⫺ supE44 relA1 deoR ⌬(lacZYA-argF)U169; Life Technologies, excess relative to FhuA.H6 prior to chromatography. Under these conditions, in
Cergy Pontoise, France], and ER2566 [F⫺ ␭⫺ fhuA2 (lon) ompT lacZ::T7 gene1 the presence of detergent, ferricrocin-iron was efficiently bound by FhuA.H6, as
gal sulA11 ⌬(mcrC-mrr)114::IS10 R(mcr-73::mini-Tn10)2 R(zgb-210::Tn10) shown by the reduction in the area of the ferricrocin-iron (Mr, 740) peak (data
endA1 (dcm); New England Biolabs, Beverly, Mass.] were used for chromosomal not shown on chromatograms). Size exclusion chromatography was performed at
DNA isolation, routine subcloning, and protein expression, respectively. Strains room temperature.
KP1060 [GM1 ⌬(ompT-fepA-entF)] (54), KP1120 [KP1060 ⌬(trp-tonB-opp- Analytical ultracentrifugation. Sedimentation velocity and sedimentation
ana)467] (22) KP1060fhuA (43), and KP1120fhuA (43) were used to evaluate the equilibrium measurements were obtained by using an AN 60-Ti rotor in a
specificity and titer of polyclonal anti-TonB chicken serum. QIAprep spin mini- Beckman XL-A analytical ultracentrifuge equipped with an optical absorbance
prep columns, a QIAquick PCR purification kit, and Ni2⫹-nitrilotriacetate system (Laboratoire d’Enzymologie, Centre National de la Recherche Scienti-
(NTA) agarose resin were purchased from Qiagen (Courtaboeuf, France); plas- fique, Gif-sur-Yvette, France). All measurements were obtained at 20°C and in
mid pET-28 was obtained from Novagen (Madison, Wis.). Restriction enzymes, 100 mM sodium phosphate, pH 7.9. The concentration of H6-⬘TonB was 0.8
Pwo thermostable proofreading DNA polymerase, and T4 DNA ligase were mg/ml. For velocity centrifugation, a rotor speed of 12,000 rpm was used. Sed-
purchased from Eurogentec (Seraing, Belgium). Antihistidine monoclonal anti- imentation equilibrium runs were performed at 12,000 and 18,000 rpm. After the
body, bromochloroindolyl phosphate, nitroblue tetrazolium, phenylmethylsulfo- final set of equilibrium scans was recorded, the rotor speed was increased to
nyl fluoride, and formaldehyde (37% solution in water) were obtained from 30,000 rpm to clear the meniscus of residual protein in order to calculate the
Sigma-Aldrich (Saint Quentin Fallavier, France). Protease inhibitor cocktail baseline absorbance and thus the corrected concentration at each radial incre-
VOL. 183, 2001 IN VITRO TonB-RECEPTOR INTERACTIONS 2757

ment. XL-A data analysis software (Beckman) was used to calculate the protein After stirring for 30 min at the ambient temperature, the samples were centri-
distributions at equilibrium. Various fitting models for single data sets were used fuged, and the resulting supernatants were dialyzed against TLN buffer to re-
to obtain best fits in which the residuals were distributed evenly around the move the EDTA. Total protein concentrations were determined by the deter-
mean, meeting normality assumptions. The partial specific volume of the protein gent-compatible bicinchoninic acid assay (55) and adjusted to 2 mg/ml with TLN
(␯៮ ) was calculated on the basis of the amino acid composition and was taken to buffer. For a typical capture assay, 5 ␮g of purified H6-⬘TonB was incubated for
be 0.7376 cm3/g. The buffer density (␳) and viscosity (␩) were taken to be 1.007 1 h at the ambient temperature with 200 ␮g of total TLN buffer-soluble mem-
g/ml and 1.04, respectively. The Rs of the protein was calculated from the brane extract containing either FepA (extract of strain ER2566) or FhuA (ex-
sedimentation coefficient (S20, w) by using the following equation: tract of strain KP1060) that in some experiments was preincubated for 10 min
with either 2 ␮g of purified colicin B (an FepA-specific protein antibiotic) or 10
Rs ⫽ M* 共1 ⫺ ␯៮ ␳兲/6␲␩Ns20,w (1) ␮g of ferricrocin-iron (an FhuA-specific iron chelate). The equivalent of 10 ␮l of
packed Ni2⫹-NTA agarose resin was added to each tube, and the samples were
incubated for 30 min at the ambient temperature. The resin was pelleted, washed
where M* is the molecular mass of the anhydrous protein and N is Avogadro’s
extensively with TLN buffer, and incubated with 20 ␮l of 100 mM EDTA in TLN
number. The frictional ratio (Rs/Rmin) was calculated as described by Tanford et

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buffer to elute the bound protein. Eluted protein samples were each mixed with
al. (56) by using the relationship: an equal volume of electrophoresis sample buffer and boiled, and 8-␮l portions
were applied to SDS–7.5% polyacrylamide gels. After transfer to nitrocellulose
Rmin ⫽ 共3M*␯៮ /4␲N兲1/3 (2) membranes, proteins were revealed by immunoblotting with monoclonal anti-
bodies directed against either FepA (a gift from M. A. McIntosh) or FhuA
Interaction assays. For small-scale solution assays we used wild-type FhuA (antibody Fhu8.3) (41). For control experiments the same protocol was used
protein that was purified by ion-exchange chromatography and chromatofocus- except that H6-⬘TonB was omitted.
ing (3) and H6-⬘TonB that was purified as described above. All incubations were
performed at room temperature. Purified FhuA (10 ␮g; 120 pmol) was incubated
RESULTS
either with a 20-fold molar excess of ferricrocin-iron or without the siderophore
in TLN buffer in a total volume of 100 ␮l for 5 min. Purified H6-⬘TonB (2.4 ␮g; Expression and purification of hexahistidine-tagged TonB.
100 pmol) was added, and incubation was continued for 1 h. To each tube 5 ␮l The tonB gene was amplified from the chromosome of E. coli
of packed Ni2⫹-NTA agarose resin that previously had been equilibrated in TLN
buffer was added. After incubation for 30 min, the resin was pelleted by centrif-
by using PCR primers designed to eliminate codons for its
ugation and washed extensively with TLN buffer. Bound protein was eluted with N-terminal 32-amino-acid hydrophobic predicted transmem-
20 ␮l of 100 mM EDTA in TLN buffer, mixed with an equal volume of dena- brane anchor (50). Vector-derived sequences appended a 20-
turing electrophoresis sample buffer, boiled for 2 min, resolved on 10% poly- amino-acid N-terminal extension, including a stretch of six
acrylamide gels, and visualized by Coomassie blue staining. Immunoblotting of
histidine residues, to the truncated TonB to facilitate purifica-
duplicate gels (data not shown) using either an antihistidine monoclonal anti-
body or anti-TonB polyclonal chicken antibodies and anti-FhuA monoclonal tion. Cell fractionation indicated that the majority of the mem-
antibody Fhu8.3 (41) confirmed the identity and integrity of each protein. brane anchorless histidine-tagged TonB protein was present in
ELISA. Microtiter plates (Nunc ImmunoSorp Delta; Life Technologies, Cergy the cytoplasm. In Ni2⫹-chelate chromatography, a major H6-
Pontoise, France) were coated with (per well) 1 ␮g (40 pmol) of H6-⬘TonB in 50 ⬘TonB peak was recovered with 100 and 150 mM imidazole
mM Tris-HCl (pH 8.0)–150 mM NaCl (TBS) containing 0.1% Triton X-100
overnight at 4°C. The wells were blocked with TBS containing 0.1% Triton
steps (Fig. 1A). Interestingly, H6-⬘TonB (Mr, 24,880) migrated
X-100, 0.1% bovine serum albumin, and 1% nonfat milk (blocking buffer). An with a relative mobility of ca. 35 kDa in SDS-polyacrylamide
overlay of 1 ␮g (12 pmol) of purified FhuA.H6 in blocking buffer, either prein- gel electrophoresis (PAGE) gels, like wild-type TonB (Mr,
cubated or not preincubated with a 20-fold molar excess of ferricrocin-iron, was 26,100; relative mobility, 36 kDa [32, 47]). The identity of
added to each well, and each plate was incubated for 1 h at 37°C. The wells were
H6-⬘TonB was confirmed by immunoblotting duplicate gels
washed with blocking buffer, and anti-FhuA monoclonal antibodies (Fhu4.1 or
Fhu8.1 as hybridoma culture supernatants diluted 10-fold in blocking buffer) with a monoclonal antibody specific for the hexahistidine tag
were added. After 1 h of incubation at 37°C followed by extensive washing, (Fig. 1B) and with polyclonal chicken antibodies specific for
primary antibodies were detected with alkaline phosphatase-conjugated anti- TonB. Despite the presence of minor contaminating proteins
mouse antibodies and para-nitrophenylphosphate. Mean background A405 values that migrated with a relative mobility of ca. 75 kDa, H6-⬘TonB
were subtracted, and the antibody reactivities (A405 values) for eight replicates
were analyzed by using the Student t test. Other controls, to which no H6-⬘TonB
represented 90 to 95% of the eluted protein, as judged from
was added before blocking and subsequent addition of FhuA.H6 or ferricrocin- Coomassie blue- or silver-stained gels. The yield of H6-⬘TonB
loaded FhuA.H6, showed background levels of antibody binding. protein was 10 mg from 500 ml of induced culture, and the
Formaldehyde cross-linking. Purified H6-⬘TonB (4 ␮g) in 100 mM sodium protein was stable for several days at room temperature and
phosphate (pH 7.9) was incubated with purified FhuA.H6 (4 ␮g) in a buffer
for several weeks at 4°C.
consisting of 100 mM sodium phosphate (pH 7.9), 100 mM NaCl, and 0.9%
octylglucoside for 30 min at the ambient temperature. In some experiments, Characterization of H6-ⴕTonB by sedimentation equilibrium
NaCl was included at a final concentration of 1 M. Formaldehyde was added to and sedimentation velocity. The state of aggregation of H6-
a concentration of 0.1% (vol/vol), and the cross-linking reaction was allowed to ⬘TonB and its molecular mass were assessed from a sedimen-
proceed for either 5 or 20 min at the ambient temperature. Samples were mixed tation equilibrium. A plot of the natural logarithm of the cor-
with electrophoresis sample buffer, heated at 60°C for 5 min, and applied to
sodium dodecyl sulfate (SDS)–7.5% polyacrylamide gels. Proteins were visual-
rected protein concentration versus the square of the radial
ized by immunoblotting by using anti-FhuA monoclonal antibodies or anti-TonB distance (Fig. 2) revealed a slight deviation from linearity, an
polyclonal chicken antibodies and alkaline phosphatase-conjugated secondary indication of minor heterogeneity in the sample. The slope of
antibodies with bromochloroindolyl phosphate and nitroblue tetrazolium. Alter- this plot yields a quantity that is formally equal to Mⴱ(1 ⫺ ␯៮ ␳),
natively, antihistidine monoclonal antibodies were used to reveal both H6-⬘TonB
where Mⴱ is the molecular mass of the anhydrous protein, ␯៮ is
and FhuA.H6 on the same blot. Formaldehyde targets lysine, cysteine, and
tyrosine and, to a lesser extent, tryptophan, histidine, aspartate, and arginine for the partial specific volume of the protein, and ␳ is the buffer
cross-linking (10). density. A molecular mass of 30 kDa was deduced from values
Capture of FepA and FhuA by H6-ⴕTonB. E. coli ER2566 (fepA⫹ fhuA) and obtained at both 12,000 and 18,000 rpm. The deviation from
KP1060 (fhuA⫹ fepA) were grown to saturation in 500 ml of L broth with 100 ␮M the theoretical molecular mass of H6-⬘TonB (24.9 kDa) led us
2,2-dipyridyl, harvested by centrifugation, and suspended in 40 ml of TBS. Cells
were lysed by sonication on ice, and the lysate was centrifuged at 15,000 ⫻ g for
to search for contributions from a protein(s) having a higher
20 min at 4°C to pellet membranes and unlysed cells. The pellet was resuspended molecular mass. Two further fittings were performed with data
in 10 ml of TBS containing 5 mM EDTA and 1% lauryldimethylamine oxide. collected at 18,000 rpm (data not shown). Both the ideal,
2758 MOECK AND LETELLIER J. BACTERIOL.

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FIG. 1. Purification of histidine-tagged TonB. Expression of H6-
⬘TonB in E. coli ER2566 was induced with IPTG. H6-⬘TonB was
purified from the soluble cell extract by Ni2⫹-chelate chromatography.
(A) Coomassie blue-stained gel. (B) Immunoblot of a duplicate gel
probed with antihistidine monoclonal antibodies. The masses of
marker proteins (in kilodaltons) are indicated on the left. Lane 1,
clarified supernatant after cell lysis; lane 2, flowthrough after binding
to Ni2⫹-NTA agarose; lanes 3 to 7, elution profiles with 50, 100, 150,
200, and 250 mM imidazole respectively.

two-independent-population model and the self-association

model were consistent with 85 to 90% of the protein being
monomeric and the remainder being dimeric. FIG. 3. Gel filtration chromatograms of purified H6-⬘TonB and
Sedimentation velocity experiments allowed determination FhuA.H6. Elution of protein from a Superose 12 column was recorded
of the sedimentation coefficient. From the value found (1.4 S) by tracing the A280. (A) H6-⬘TonB (270 ␮g) in 100 mM sodium phos-
phate (NaPi), pH 7.9. (B) H6-⬘TonB (250 ␮g) in detergent-containing
we calculated (equation 1) that the Rs for the H6-⬘TonB pro- TLN buffer. (C) FhuA.H6 (200 ␮g) in TLN buffer.
tein was 3.8 nm. A value of 1.9 nm for Rmin was calculated with
equation 2. The value found for the frictional ratio (Rs/Rmin),
2.0, suggests that H6-⬘TonB is rather elongated. Similar calcu-
lations were done assuming a hydration level of 0.3 g of H2O/g
of protein (45), and these calculations led to a value for Rmin of
2.1 nm and a value for the frictional ratio of 1.8. Data were best
fit to a hydrated cylinder model with a length of 24 nm and a
diameter of 1.6 nm, corresponding to an axial ratio (a/b) of 15.
Size exclusion chromatography of H6-ⴕTonB. Gel filtration
analysis of purified H6-⬘TonB in phosphate buffer revealed a
single well-defined A280 peak that eluted at 12.1 ml (Fig. 3A).
The gel filtration elution profile of purified H6-⬘TonB in the
detergent-containing TLN buffer, which was used later in in-
teraction studies with FhuA, was markedly different (Fig. 3B);
H6-⬘TonB eluted as a major A280 peak (80% of the total peak
area) at 12.5 ml and as a minor broad peak (15% of the total
peak area) centered at 10.8 ml. The column was calibrated with
proteins having known Rs, and the retention volumes of these
FIG. 2. Sedimentation equilibrium of H6-⬘TonB. Solutions of H6- proteins were not significantly different whether the column
⬘TonB (0.8 mg/ml in 100 mM sodium phosphate, pH 7.9) were centri- had been equilibrated in phosphate buffer or in TLN buffer.
fuged overnight at 18,000 rpm at 20°C. The corrected protein concen- The Rs of H6-⬘TonB was estimated from the calibration curve
tration, (C) was calculated from the A280 and was plotted as a function
of distance from the center of rotation (r). The molecular mass of
of Rs versus KD; the value in phosphate buffer was 4.3 nm,
H6-⬘TonB was estimated by fitting the concentration gradients to a whereas the major A280 peak in TLN buffer corresponded to an
single-ideal-component model. Rs of 4.1 nm. From the known sedimentation coefficient value
VOL. 183, 2001 IN VITRO TonB-RECEPTOR INTERACTIONS 2759

Quantitation of ligand-induced formation of the H6-ⴕTonB–

FhuA.H6 complex. An ELISA was developed to better quantify
the interaction between FhuA and TonB. H6-⬘TonB was ad-
sorbed to wells of a microtiter plate, the remaining protein
binding sites were blocked, and a second FhuA.H6 or
FhuA.H6-ferricrocin-iron antigen layer was added. After wash-
ing, bound FhuA.H6 was detected with anti-FhuA monoclonal
antibodies Fhu4.1 and Fhu8.1 (41), each of which recognizes a
different surface-exposed epitope of FhuA. The absorbance
values were at least threefold higher for the FhuA.H6-ferric-
FIG. 4. Capture of purified wild-type FhuA by H6-⬘TonB is pro-
rocin-iron complex than they were for FhuA.H6 alone; with
moted by ferricrocin. H6-⬘TonB was incubated with FhuA or with

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ferricrocin-loaded FhuA, and complexes were captured with Ni2⫹- antibody Fhu4.1, the A405 without ferricrocin-iron was 0.65 ⫾
NTA agarose. Proteins were eluted from the resin, resolved by SDS- 0.18 (mean ⫾ standard deviation; n ⫽ 8), whereas with the
PAGE, and stained with Coomassie blue. The masses of marker pro- ligand present the A405 increased by more than 200% to 2.04 ⫾
teins (lane M) (in kilodaltons) are indicated on the left. Lane T, 400 ng 0.11. Similarly, with antibody Fhu8.1 the A405 without ferric-
of H6-⬘TonB; lane F, 200 ng of wild-type FhuA; lane 1, control eluate
from incubation of H6-⬘TonB without FhuA; lane 2, control eluate rocin-iron was 0.42 ⫾ 0.07, while in the presence of the ligand
from incubation of FhuA-ferricrocin without H6-⬘TonB; lane 3, eluate the A405 was 1.75 ⫾ 0.17. As assessed by Student’s t test, the
from incubation of H6-⬘TonB with FhuA; lane 4, eluate from incuba- differences in the mean absorbance values for the eight repli-
tion of H6-⬘TonB with FhuA-ferricrocin. cates were highly significant (P ⬍ 10⫺5). Since the reactivities
of anti-FhuA monoclonal antibodies Fhu4.1 and Fhu8.1 are
independent of the presence of ferricrocin-iron bound to the
(1.4 S), we estimated the molecular mass of H6-⬘TonB using receptor (42), we concluded that ferricrocin-iron substantially
equation 1 (see Materials and Methods). The values found (28 increased the association between FhuA and TonB. A second
kDa in phosphate buffer, 26 kDa in TLN buffer) are in rea- ELISA, in which FhuA.H6 (either with or without pretreat-
sonable agreement with the molecular masses of H6-⬘TonB ment with ferricrocin-iron) was used to coat the microtiter
calculated from its sequence (24.9 kDa) and determined by plate, H6-⬘TonB was the second layer, and bound H6-⬘TonB
sedimentation equilibrium (30 kDa). Determination of the Rs was detected by anti-TonB polyclonal chicken antibodies, pro-
and molecular mass of the species corresponding to the minor duced similar results.
peak that eluted in TLN buffer suffered from uncertainty due Purified H6-ⴕTonB and FhuA.H6 proteins form a formalde-
to the broadness of the peak. hyde-cross-linked complex in vitro. Previous studies identified
Purified H6-ⴕTonB and wild-type FhuA interact in solution. complexes between TonB and FepA (54) and between TonB
A small-scale solution assay was developed to investigate and FhuA (43) that were formed in vivo and were stabilized by
whether purified H6-⬘TonB could interact with FhuA, a pro- formaldehyde cross-linking. We performed a formaldehyde-
totypic TonB-dependent receptor of E. coli. A previous study cross-linking experiment after allowing H6-⬘TonB and
demonstrated that purified histidine-tagged FhuA (FhuA.H6) FhuA.H6 to interact in solution in an effort to estimate the
(42) bound wild-type TonB from detergent-solubilized cell ex- molecular mass of the complex. FhuA.H6 remained exclusively
tracts and that the resulting complex could be captured from monomeric in the presence of 0.1% formaldehyde (Fig. 5A).
solution by using Ni2⫹-NTA agarose resin (43). Similar assays While purified H6-⬘TonB remained monomeric following in-
performed with H6-⬘TonB, therefore, necessitated the use of cubation with formaldehyde (Fig. 5B, compare lanes 1 and 2),
purified wild-type FhuA (3) so that only one of the two part- the protein showed a slight tendency to oligomerize into com-
ners possessed the hexahistidine tag. By a number of criteria, plexes with apparent mobilities between 70 and 80 kDa as
including antibody reactivity, ligand binding in vitro and in visualized by immunoblotting of overloaded gels (data not
vivo, and three-dimensional structure (17, 37, 42), wild-type shown). Incubation of the purified proteins together using an
FhuA and FhuA.H6 are structurally and functionally equiva- approximately threefold molar excess of H6-⬘TonB over FhuA
lent. After incubation of FhuA and H6-⬘TonB in vitro, com- led to recruitment of a fraction of each protein into a complex
plexes were captured with Ni2⫹-NTA agarose and examined by with a molecular mass of ca. 150 kDa (Fig. 5A and B, lane 6)
SDS-PAGE. While capture of FhuA by H6-⬘TonB apparently as revealed by immunoblotting with FhuA- and TonB-specific
required prior binding of ferricrocin-iron to FhuA (Fig. 4, antibodies. This finding can be considered in agreement with
compare lanes 3 and 4), overloading of the gels (data not the relative mobility of the complex as determined after form-
shown) allowed visualization of small amounts of FhuA that aldehyde cross-linking in vivo (43), allowing for a certain de-
were recovered without addition of the siderophore. The gree of error given (i) the imprecision of relating relative
FhuA-ferricrocin-iron complex that was incubated under the mobility to molecular mass for unboiled cross-linked samples
same conditions but in the absence of H6-⬘TonB showed no and (ii) the demonstrated aberrant migration of TonB and
affinity for the resin (Fig. 4, lane 2), demonstrating the speci- H6-⬘TonB in polyacrylamide gels. Preincubation of FhuA with
ficity of the capture. Since colicin B, an FepA-specific protein ferricrocin-iron increased slightly the abundance of the TonB-
antibiotic, was unable to promote the FhuA–H6-⬘TonB inter- FhuA complex without altering its apparent molecular mass
action (data not shown), the small-scale assay therefore estab- (Fig. 5A and B, lane 8).
lished that purified H6-⬘TonB formed a specific complex with The cross-linking results demonstrated that FhuA formed a
FhuA since complex formation was greatly augmented by prior complex with TonB even in the absence of an FhuA-specific
binding of ferricrocin-iron to the receptor. ligand. This result apparently conflicted with the results of the
2760 MOECK AND LETELLIER J. BACTERIOL.

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FIG. 6. Ferricrocin stabilizes the FhuA-TonB complex against high
salt concentrations. Samples of H6-⬘TonB and FhuA.H6 were mixed in
the presence or absence of 1 M NaCl as indicated at the bottom, and
then formaldehyde was added to all samples. Cross-linking was al-
lowed to proceed for 20 min (lanes 1 to 4) or 5 min (lanes 5 to 8). Equal
volumes of aliquots were subjected to SDS-PAGE, and H6-⬘TonB and
FhuA.H6 proteins were visualized simultaneously by immunoblotting
with antihistidine monoclonal antibodies. The masses of marker pro-
teins (in kilodaltons) are indicated on the left. The samples in lanes 2,
4, 6, and 8 contained 1 M NaCl; the samples in lanes 3, 4, 7, and 8 were
incubated in the presence of the FhuA-specific ligand ferricrocin prior
to cross-linking.

to the monomeric form of FhuA.H6 plus its detergent belt

(total Mr, 185,000). The presence of ferricrocin-iron had no
effect on the elution profile of FhuA.H6 or H6-⬘TonB (data not
shown). It should be noted that the A280 maximum of the
H6-⬘TonB peak (Fig. 3A and B [270 and 250 ␮g of H6-⬘TonB
FIG. 5. Formaldehyde cross-linking traps the FhuA-TonB com- applied, respectively]) was markedly lower than the A280 max-
plex. Samples of H6-⬘TonB were mixed with FhuA.H6, and formalde- imum of the FhuA.H6 peak (Fig. 3C [200 ␮g of FhuA.H6
hyde was added to some samples as indicated at the bottom. Aliquots
were resolved on duplicate SDS–7.5% polyacrylamide gels, and pro-
applied]). Protein concentrations were determined carefully
teins were visualized by immunoblotting with either anti-FhuA mono- prior to chromatography (55) and were validated by estimation
clonal antibody Fhu4.1 (A) or anti-TonB polyclonal chicken antibodies from both Coomassie blue- and silver-stained polyacrylamide
(B). The masses of marker proteins (in kilodaltons) are indicated on gels with protein standards. The presence of a single trypto-
the left. Lanes 1 and 2, H6-⬘TonB alone; lanes 3 and 4, FhuA.H6 alone; phan residue in H6-⬘TonB compared to nine tryptophan resi-
lanes 5 and 6, FhuA.H6 plus H6-⬘TonB; lanes 7 and 8, FhuA.H6-
ferricrocin plus H6-⬘TonB. Lanes 1, 3, 5, and 7 contained untreated dues in FhuA.H6 may in part explain the relatively weak A280
samples; the samples in lanes 2, 4, 6, and 8 were incubated with 0.1% of H6-⬘TonB.
formaldehyde for 20 min prior to electrophoresis. Addition of increasing amounts of H6-⬘TonB relative to
FhuA.H6 in the absence of ferricrocin-iron did not significantly
shift the position of the eluting peak (Fig. 7), which corre-
solution capture assay which demonstrated the need for ligand sponded well to the pure FhuA.H6 peak (11.6 ml) (Fig. 3C). In
occupation of FhuA for formation of significant amounts of a marked contrast, addition of increasing amounts of H6-⬘TonB
complex with TonB. We addressed this apparent discrepancy to ferricrocin-loaded FhuA.H6 progressively shifted the A280
by cross-linking in the presence of a high concentration of salt peak to 10.7 ml (Fig. 7). At equimolar ratios of H6-⬘TonB and
to increase the stringency of the interaction environment. ferricrocin-loaded FhuA (Fig. 7C), the peak at 10.7 ml ac-
While under stringent conditions in the absence of the ligand counted for more than 96% of the total peak area. SDS-PAGE
the efficiency of formaldehyde cross-linking was greatly re- analysis (data not shown) confirmed the identities of FhuA.H6
duced (Fig. 6, compare lanes 1 and 2 and lanes 5 and 6), the and H6-⬘TonB in the eluates and their shift to earlier fractions
ferricrocin-loaded FhuA–H6-⬘TonB complex was stable in the when ferricrocin was present; even at the lowest molar ratio of
presence of 1 M NaC1 (Fig. 6, lanes 4 and 8). H6-⬘TonB to ferricrocin-loaded FhuA (10-fold molar excess of
Characterization of H6-ⴕTonB–FhuA.H6 complexes by size FhuA) (Fig. 7A), H6-⬘TonB was found exclusively in fractions
exclusion chromatography. Since results from the gel filtration corresponding to the earliest elution volumes. These results
experiments were in good agreement with the sedimentation are best explained by ferricrocin-induced formation of a com-
data, size exclusion chromatography was used to further char- plex between FhuA.H6 and H6-⬘TonB. The Rs of the complex,
acterize the H6-⬘TonB–FhuA.H6 complex. Purified FhuA.H6 as deduced from the calibration curve relating Rs to KD, was 6.2
eluted in a major A280 peak at 11.6 ml (Fig. 3C). The value nm. Rmin was calculated by using equation 2 (see Materials and
obtained for its Rs (4.8 nm) is close to that determined for Methods); in this analysis the partial specific volume of FhuA
wild-type FhuA in octylglucoside-containing buffer (4.5 nm) was taken to be 0.776 cm3/g (3) and it was assumed that
(3). It is therefore highly likely that the A280 peak corresponds H6-⬘TonB bound to FhuA in its detergent-bound form (total
VOL. 183, 2001 IN VITRO TonB-RECEPTOR INTERACTIONS 2761

FIG. 8. H6-⬘TonB captures FepA and FhuA. Purified H6-⬘TonB

was incubated with cell extracts containing either wild-type FepA or
FhuA. Complexes were captured with Ni2⫹-NTA agarose, resolved by

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SDS-PAGE, and immunoblotted with a monoclonal antibody against
FepA (A) or FhuA (B). (A) Lane 1, 15 ␮g of total soluble ER2566
extract; lane 2, control without added H6-⬘TonB; lane 3, control with-
out added H6-⬘TonB but with colicin B added; lane 4, control eluate
without added ER2566 cell extract; lane 5, control eluate without
added ER2566 cell extract but with colicin B added; lane 6, eluate from
incubation of H6-⬘TonB with ER2566 cell extract; lane 7, eluate from
incubation of H6-⬘TonB with ER2566 cell extract preincubated with
colicin B. (B) Lane 1, 100 ng of FhuA.H6 (migration standard); lane 2,
15 ␮g of total soluble KP1060 extract; lane 3, negative control capture
experiment without added H6-⬘TonB; lane 4, control eluate without
added KP1060 cell extract; lane 5, control eluate without added
KP1060 cell extract but with ferricrocin-iron added; lane 6, eluate from
incubation of H6-⬘TonB with KP1060 cell extract; lane 7, eluate from
incubation of H6-⬘TonB with KP1060 cell extract preincubated with
ferricrocin-iron.

FepA-specific ligand colicin B [48]) were incubated with H6-

⬘TonB. Complexes were captured with Ni2⫹-NTA agarose and
identified by immunoblotting with an anti-FepA monoclonal
antibody. FepA was detected as a single band at ca. 80 kDa in
the eluate (Fig. 8A). Formation of the complex between FepA
and H6-⬘TonB was specific since pretreatment with the FepA-
specific ligand colicin B increased substantially the abundance
of the FepA–H6-⬘TonB complex (Fig. 8A, compare lanes 6 and
7) and since FepA alone showed no affinity for the resin in the
absence of H6-⬘TonB (Fig. 8A, lanes 2 and 3 [negative con-
FIG. 7. Analysis of H6-⬘TonB–FhuA.H6 interaction by gel filtra- trols]). A control experiment confirmed that wild-type FhuA,
tion. Proteins were incubated together at various molar ratios and then also expressed from the chromosome, was bound by H6-⬘TonB;
applied to a Superose 12 column. Dotted lines, experiments conducted
without ferricrocin; solid lines, ferricrocin added to the protein sample capture of wild-type FhuA was greatly enhanced by preincu-
before it was applied to the column. In all experiments, the amount of bation of wild-type FhuA with ferricrocin-iron (Fig. 8B, lanes
FhuA.H6 applied to the column was 200 ␮g; the amount of H6-⬘TonB 6 and 7) or with a crude cell extract containing the FhuA-
ranged from 5 ␮g (ca. 10-fold molar excess of FhuA) (A) to 18 ␮g (ca. specific protein antibiotic colicin M (data not shown).
3-fold molar excess of FhuA) (B) to 60 ␮g (the concentrations of TonB
and FhuA were approximately equal) (C).
The possibility that FepA and FhuA bound to H6-⬘TonB
indirectly via colicin M and colicin B was discounted by a
control experiment (Moeck, unpublished data) which demon-
strated that colicin killing activity against susceptible E. coli
Mr, 185,000) (3). An Rmin value was calculated by assuming strains in vivo was independent of prior incubation of the
that the H6-⬘TonB–FhuA.H6 molar stoichiometry was 1/1 with colicin solutions (colicin B purified protein, colicin M colici-
both proteins hydrated. With this value (Rmin ⫽ 4.4), we esti- nogenic cell extract) with H6-⬘TonB and removal of any puta-
mated that the frictional ratio (Rs/Rmin) was 1.4, a value sig- tive TonB-colicin complexes by precipitation with Ni2⫹-NTA
nificantly different from and between the frictional ratio values agarose.
of H6-⬘TonB (1.8) and FhuA (1.2); the latter value is typical for
a globular protein (3). DISCUSSION
Capture of FepA by histidine-tagged TonB. We hypothe-
sized that H6-⬘TonB could bind not only to FhuA but also to The work of Jaskula et al. (22) demonstrated that an an-
other TonB-dependent receptors. The ferric enterobactin re- chorless form of TonB, although failing to energize TonB-
ceptor FepA was chosen to test this hypothesis. E. coli cells dependent processes in vivo, remained able to form a complex
expressing wild-type FepA from the chromosome were lysed, with the ferric enterobactin receptor FepA. Experiments with
the membranes were solubilized in detergent-containing the purified ferrichrome-iron receptor from E. coli indicated
buffer, and portions (pretreated or not pretreated with the that wild-type TonB, solubilized from the cytoplasmic mem-
2762 MOECK AND LETELLIER J. BACTERIOL.

brane, was capable of both binding the receptor and sensing its that H6-⬘TonB has an elongated form with an axial ratio of 15,
ligand occupation status (43). These results demonstrated that so it may take the form of a rod that is 24 nm long and 1.6 nm
the interaction between receptors and both wild-type and N- in diameter.
terminally truncated TonB proteins could occur in the absence These observations are reminiscent of recent data for the
of the proton motive force and thus promoted the feasibility of AcrA protein of the multidrug efflux complex of E. coli and for
in vitro reconstitution of high-affinity iron transport. To ad- the TolA protein of the group A colicin import system of E.
vance studies in this area, we isolated a TonB variant that coli. These two proteins share features with TonB; they are
lacked the N-terminal signal anchor. The favorable yield, pu- anchored in or associated with the cytoplasmic membrane, and
rity, and stability of H6-⬘TonB make it a good candidate for they are thought to extend into and across the periplasm. AcrA
crystallization and structure determination. Knowledge of the adopts a highly asymmetric form, as assessed by dynamic light
three-dimensional structure of TonB is essential for a complete scattering and analytical ultracentrifugation (59). This finding

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understanding of bacterial iron nutrition and thus for designing is consistent with the presence of a 75-amino-acid central re-
iron transport inhibitors with potential therapeutic value gion of AcrA that is predicted to be largely ␣-helical. This
against bacterial infections. region may span a distance of 10 nm, thereby aiding in the
The homogeneity of the H6-⬘TonB preparation was ascer- formation of protein-protein contacts (60) across the cell en-
tained by sedimentation equilibrium experiments. The data velope. Similarly, the central ␣-helical domain of TolA adopts
were consistent with 85 to 90% of the protein being mono- a rigid form (14, 36), probably allowing the protein to span the
meric and the remainder being dimeric. Size exclusion chro- periplasm to contact group A colicins, including colicins A and
matography of H6-⬘TonB in phosphate buffer was indicative of E1 (4, 15, 33). Based on these results and in agreement with a
a monodisperse and monomeric preparation, while 10 to 15% nuclear magnetic resonance study of a synthetic peptide cor-
dimers were observed in another preparation of H6-⬘TonB (in responding to the central proline-rich region of TonB (9), we
detergent-containing TLN buffer). Cross-linking experiments propose that the elongated form of H6-⬘TonB reflects the form
indicated that H6-⬘TonB had a slight tendency to form oli- adopted by wild-type TonB and may bridge the cytoplasmic
gomers with apparent mobilities between 70 and 80 kDa. and outer membranes, which are thought to be separated by 15
These results are supported by the presence of H6-⬘TonB oli- to 20 nm (19).
gomers, as seen in mass spectrometry analysis (G. S. Moeck Size exclusion chromatography, ELISA, and in vitro capture
and B. Thatcher, unpublished data). A 77-kDa TonB-contain- experiments all showed that TonB-FhuA contact was greatly
ing complex was observed in previous studies of in vivo cross- enhanced by prior binding of ferricrocin-iron to FhuA. Form-
linking between TonB and other proteins of the cell envelopes aldehyde cross-linking stabilized a high-molecular-mass com-
of E. coli (54) and other gram-negative bacteria (30); however, plex (relative mobility, ca. 150 kDa) that was detected by both
the composition of the 77-kDa complex remains unknown. anti-FhuA and anti-TonB antibodies. Preliminary results from
Taking into account the retarded migration of TonB in SDS- mass spectrometry analysis (Moeck and Thatcher, unpub-
PAGE gels, it is likely that the 70- to 80-kDa complex which lished) indicate that the stoichiometry of the complex corre-
was observed in the present study represents dimers of H6- sponds to that of a H6-⬘TonB–FhuA.H6 heterodimer, equiva-
⬘TonB. In this regard, the native TonB amino acid sequence lent to the stoichiometry observed for the site-directed
shows a weak propensity to form coiled coils (38) via interac- cysteine-cross-linked BtuB-TonB heterodimer (relative mobil-
tions in a 14-amino-acid window (residues A199 to R212) near ity, 100 kDa) (12). By comparison, previous studies of in vivo
the C terminus. A ␤-strand–␣-helix–␤-strand motif involving cross-linking (30, 54) revealed a TonB-FepA complex with a
this region has been proposed for TonB (26, 28). relative mobility of ca. 195 kDa. Mass spectrometry and ana-
Like wild-type TonB, H6-⬘TonB showed anomalous behav- lytical ultracentrifugation using H6-⬘TonB may now be ex-
ior in polyacrylamide gels. It migrated with a relative mobility ploited to confirm the masses and stoichiometry of the various
of ca. 35 kDa, which is similar to the apparent molecular mass TonB-receptor complexes in a manner that is independent of
of wild-type TonB (35 to 36 kDa in SDS-PAGE gels [29, 47]) their molecular forms. Furthermore, surface plasmon reso-
and is considerably different than would be predicted from its nance techniques may now permit kinetic analyses of the in-
actual molecular mass of 25 kDa as deduced from its nucleo- teractions between TonB and purified TonB-dependent recep-
tide sequence. It was concluded that the aberrant mobility of tors, as demonstrated for the interactions between fragments
wild-type TonB stems from its characteristic proline-rich re- of the E. coli TolA protein, a structural analogue of TonB, and
gion since a TonB derivative carrying a deletion of this region the colicins which it translocates (14).
(TonB⌬66-100) had a relative electrophoretic mobility in poly- Two independent experiments indicated that H6-⬘TonB in-
acrylamide gels that reflected its actual molecular mass (28). teracted in vitro with FhuA even in the absence of ferricrocin-
By inference, the aberrant migration of H6-⬘TonB indicates iron. The H6-⬘TonB–FhuA complex was observed in Ni2⫹-
that its proline-rich region adopts a conformation which is NTA agarose capture experiments after the gel was overloaded
similar to that of wild-type TonB. and the H6-⬘TonB–FhuA.H6 complex was visualized after
H6-⬘TonB also behaved anomalously in gel filtration studies cross-linking of the two proteins. However, no evidence for
since its migration was delayed compared to that expected for formation of a complex in the absence of ferricrocin-iron was
a globular soluble protein with the equivalent molecular mass. obtained by size exclusion chromatography. These results may
Anomalous elution through gel filtration matrices has been reflect a weak affinity of TonB for its receptors in the absence
described for elongated proteins, such as myosin and fibrino- of their cognate ligands, as previously noted (43, 54). Such
gen (44). The combination of size exclusion chromatography weak TonB-receptor interactions are likely to be overrepre-
and sedimentation velocity measurements indeed indicated sented by formaldehyde cross-linking since in the present study
VOL. 183, 2001 IN VITRO TonB-RECEPTOR INTERACTIONS 2763

they were selectively disrupted with high concentrations of salt problem. Trends Biochem. Sci. 24:104–109.
8. Braun, V., K. Hantke, and W. Köster. 1998. Bacterial iron transport: mech-
in the absence of an FhuA-specific ligand. anisms, genetics, and regulation, p. 67–145. In A. Sigel and H. Sigel (ed.),
In vitro experiments with H6-⬘TonB realistically may now be Metal ions in biological systems, vol. 35. Iron transport and storage in
extended to further define the regions of contact between microorganisms, plants, and animals. Marcel Dekker, New York, N.Y.
9. Brewer, S., M. Tolley, I. P. Trayer, G. C. Barr, C. J. Dorman, K. Hannavy,
TonB and TonB-dependent receptors. The TonB box (51), a C. F. Higgins, J. S. Evans, B. A. Levine, and M. R. Wormald. 1990. Structure
short stretch of sequence homology near the N termini of and function of X-Pro dipeptide repeats in the TonB proteins of Salmonella
high-affinity outer membrane receptors, is one region of TonB- typhimurium and Escherichia coli. J. Mol. Biol. 216:883–895.
10. Brinkley, M. 1992. A brief survey of methods for preparing protein conju-
receptor contact. Site-directed disulfide cross-linking revealed gates with dyes, haptens, and cross-linking reagents. Bioconjug. Chem. 3:2–
close associations between residues in the TonB box of BtuB 13.
11. Buchanan, S. K., B. S. Smith, L. Venkatramani, D. Xia, L. Esser, M. Palnit-
and residues around amino acid 160 of TonB (12, 13). Crystallo- kar, R. Chakraborty, D. van der Helm, and J. Deisenhofer. 1999. Crystal
graphic analyses revealed dramatic ligand-induced changes on structure of the outer membrane active transporter FepA from Escherichia

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the periplasmic face of FhuA (17, 37), which occurred just C coli. Nat. Struct. Biol. 6:56–63.
12. Cadieux, N., C. Bradbeer, and R. J. Kadner. 2000. Sequence changes in the
terminal to the TonB box of FhuA, which is located between Ton box region of BtuB affect its transport activities and interaction with
amino acids 7 and 11. Similarly, the TonB box of BtuB under- TonB protein. J. Bacteriol. 182:5954–5961.
went a striking rearrangement upon addition of Vitamin B12 to 13. Cadieux, N., and R. J. Kadner. 1999. Site-directed disulfide bonding reveals
an interaction site between energy-coupling protein TonB and BtuB, the
outer membrane preparations of E. coli; its helical conforma- outer membrane cobalamin transporter. Proc. Natl. Acad. Sci. USA 96:
tion was converted into an extended, disordered structure that 10673–10678.
14. Derouiche, R., R. Lloubès, S. Sasso, H. Bouteille, R. Oughideni, C. Lazdun-
may have extended into the periplasm (39). The TonB box of ski, and E. Loret. 1999. Circular dichroism and molecular modeling of the E.
FepA (residues 12 to 18) was sufficiently ordered in FepA coli TolA periplasmic domains. Biospectroscopy 5:189–198.
crystals (11) to be modeled; however, its extended conforma- 15. Derouiche, R., G. Zeder-Lutz, H. Bénédetti, M. Gavioli, A. Rigal, C. Laz-
dunski, and R. Lloubès. 1997. Binding of colicins A and E1 to purified TolA
tion and ambiguity about the ligand occupation status of the domains. Microbiology 143:3185–3192.
receptor in the crystal revealed little in terms of a distinct 16. Evans, J. S., B. A. Levine, I. P. Trayer, C. J. Dorman, and C. F. Higgins. 1986.
structure that might be sensed by TonB. The ligand-mediated Sequence imposed structural constraints in the TonB protein of Escherichia
coli. FEBS Lett. 208:211–216.
conformational changes in FhuA and FepA that resulted in 17. Ferguson, A. D., E. Hofmann, J. W. Coulton, K. Diederichs, and W. Welte.
increased coupling with TonB, as manifested in the present 1998. Siderophore-mediated iron transport: crystal structure of FhuA with
work, may be closely related to those revealed by crystallogra- bound lipopolysaccharide. Science 282:2215–2220.
18. Fischer, E., K. Günter, and V. Braun. 1989. Involvement of ExbB and TonB
phy. Structural analyses of TonB-receptor cocrystals are nec- in transport across the outer membrane of Escherichia coli: phenotypic
essary to define with precision the protein-protein contact sur- complementation of exb mutants by overexpressed tonB and physical stabi-
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