Exchangeability of N-Termini in the Ligand-Gated Porins of

Escherichia coli

Daniel C. Scott, Zhenghua Cao, Zengbiao Qi, Matthew Bauler, John D. Igo,

Salete M. C. Newton & P. E. Klebba*

Department of Chemistry & Biochemistry

University of Oklahoma

Norman, OK 73019

Running title: Exchange of LGP N-termini

* Corresponding author: peklebba@ou.edu

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Summary

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest an unique

architecture: their N-termini fold into a globular domain that lodges within, and physically obstructs, a

transmembrane porin $-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, in spite of their aberrant or missing N-termini, confirming (Braun et al., Mol.

Microbiol. 33:1037-1049, 1999) 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.

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Introduction

Gram-negative bacteria recognize and transport ferric siderophores (1,2) through proteins in their outer

membrane (OMa). 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 who 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 Haemophilus (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 $-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 transport into the cell (33-35). In this sense the receptors are ligand-gated. Finally, LGP transport

a Abbreviations: OM, outer membrane; LGP, ligand-gated porin; FeEnt, ferric enterobactin; Fc, ferrichrome; MAb, monoclonal antibody; SDS, sodium dodecyl sulfate; LDS, lithium dodecyl sulfate; MOPS, morphilino propane sulfonic acid; PCR,

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requires proton motive force (36-38) and the participation of another cell envelope protein, TonB (39-41),

that 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.

Their ligand recognition and transport processes, for example, are only vaguely defined. The OM

receptors of Escherichia coli discriminate numerous ferric siderophore complexes, that in spite of 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), while 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, that 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.

Experimental Procedures

Bacterial Strains, Plasmids and media. Bacteria harboring plasmids (Table 1) carrying the genes of

interest were cultured in LB broth or MOPS minimal media (46). It was impractical to create single-copy

polymerase chain reaction; TBS, tris-buffered saline; SulfoEGS, ethylene glycobis(sulfosuccimidylsuccinate).

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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 2-3 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$, we PCR-amplified 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 Fhu$, flanked by BamHI and SacI restriction sites, and joined it

to FepN in pHSG575. Additionally, we employed oligonucleotide-directed mutagenesis (Quik Change,

Strategize Corp., San Diego) 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.

Binding and transport. The adsorption of 59FeEnt and 59FeFc was measured with metabolically

inactive KDF541 (46) expressing FepA or FhuA, respectively. 59FeEnt and 59FeFc were prepared at a

specific activity of approximately 200 cpm/pM and chromatographically purified. Binding manipulations

were performed at 0o C. A mid-log bacterial culture was chilled on ice for 1 hour and an aliquot

(containing approximately 5 x 107 cells), was pipetted into a 50 ml test tube and incubated on ice. A 25

ml volume of ice cold MOPS minimal media, containing varying concentrations of 59Fe-siderophore, was

poured into the tube to achieve rapid and thorough mixing. After 1 minute the binding reactions, which

were performed in triplicate, were filtered through 0.45 : nitrocellulose, the filters were were washed

with 10 ml of 0.9% LiCl and counted in a Packard Cobra gamma counter. The initial adsorption reaction

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reached equilibrium within 5 s at physiological temperatures, and within 1 minute on ice. The FepA-

deficient strain KDF541 was simultaneously tested as a negative control, and any non-specific adsorption

of 59FeEnt 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 minutes) to

decrease standard error. Binding data were analyzed using the Bound vs Total equation of Grafit 4.013

(Erithacus Ltd., Middlesex, UK).

Ferric siderophore uptake was qualitatively evaluated by nutrition tests (44), and the transport of

59FeEnt and 59FeFc 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). The 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 describes the

outer membrane components of the process, FepA, FhuA, and their mutant derivatives.

Transport manipulations were performed at 37o C. A volume of mid-log bacterial culture (50 - 100

:l containing approximately 5 x 107 cells) was pipetted into a 50 ml test tube and incubated in a 37o C

water bath. Without delay, 25 ml of pre-warmed MOPS minimal media, containing glucose (0.2%),

appropriate nutritional supplements and varying concentrations of 59FeEnt, was poured into the tube to

achieve rapid and thorough mixing. The transport reactions were quenched by the addition of a 1,000-fold

excess of non-radioactive FeEnt, immediately filtered through 0.45 : nitrocellulose, 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 FeEnt uptake, which were calculated at each substrate

concentration from two independent measurements made in triplicate at 5 s and 15 s: cpm bound to the

cells at 5s were subtracted from the cpm associated with the cells at 15s (10 s uptakes). For some mutants

with low uptake rates (FepNFhuβ, FhuNFepβ, Fepβ) we extended the uptake period to 60 minutes (6). In

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all transport experiments the FepA-deficient strain KDF541 was simultaneously tested as a negative

control, and any non-specific adsorption of 59FeEnt by this strain was subtracted from the experimental

samples. Transport results were analyzed according to the Michaelis-Menten equation, using Grafit 4.013.

Bacteriocin and bacteriophage susceptibility. Serial dilutions of colicins B, D or M, and bacteriophage

T5 and N80 were prepared in LB broth in microtiter plates, and 5 ul 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.

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 ug of OM or 2 ug of purified protein was resuspended in LDS

sample buffer and sonicated in an ice water bath for five minutes. After a 1 min centrifugation in a

microfuge, the samples were electrophoresed overnight at 5 mA and 4 oC 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

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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 ug/ml and 0.5

ug/ml, respectively, dispensed into microtiter plates (Immulon), and incubated at 4 oC overnight. All

further incubation steps were performed at 25 oC. In the morning, excess or unabsorbed antigen was

removed by 3 washes with Tris-buffered saline containing 0.05% Tween 20, pH 7.4 (TBS-tween), and

the plates were blocked with 2% BSA in TBS for 30 min. The plates were washed 3 times with TBS-

tween, and serial dilutions of anti-FepA MAbs in 50 µl of blocking buffer were added and incubated for

1 hour. The blocking buffer was removed and 50 µl of a 1/100 dilution of goat-anti-mouse

immunoglobulin-alkaline phosphatase (Sigma) in blocking buffer was added to the plates. After

incubation for 1 hour at 25 oC, the plates were washed 3 times and developed with 50 ul of p-nitrophenyl

phosphate (1mg/ml; Sigma). After a 1 hour incubation at 25 oC, 50 µl of 2N NaOH was added to stop the

reaction and absorbance was measured at 405 nm with a microplate reader.

OM protein crosslinking. 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 crosslinker (mol. wt. 661 D) that preferentially reacts with primary amines, for 2 hours

at 0 oC. The compound contains a 16Å spacer arm, that is cleaved by reaction with hydroxylamine. After

crosslinking, cells or OM proteins were solubilized in sample buffer, subjected to SDS-PAGE, and stained

with coomassie blue. When indicated, 5 uM ferric enterobactin was added to the cells prior to

crosslinking. Crosslinked bands were excised from the gels, cleaved with hydroxylamine, electroeluted

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and re-electrophoresed: the identity of the crosslinked proteins was determined by sequence analysis of

their N-terminal 15 residues (Protein and Nucleic Acid Sequence Facility, Medical College of Wisconsin).

Results

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 $-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.

The hybrid proteins functioned with unexpected efficiency and selectivity, conferred by the $-

barrels and their loops. The N-terminal domains did not affect the discrimination of ferric siderophores.

FhuNFep$ bound FeEnt but not ferrichrome, whereas FepNFhu$ bound ferrichrome but not FeEnt. FepA

adsorbs FeEnt with sub-nanomolar affinity, that perservered in the FhuNFep$ chimera: the Kd of its

binding reaction with FeEnt was 0.2 nM (Fig 2, Table 2). The reverse hybrid (FepNFhu$) bound

ferrichrome with equivalent affinity to that of wild type FhuA: the Kd its binding reaction with Fc was

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0.6 nM (Fig 2, Table 2). So, in spite of their heterologous N-termini, the chimeras maintained the

selectivity of their $-barrels, with the affinity of wild type receptors.

The constructs that deleted the N-domains of FepA and FhuA (Fep$ and Fhuβ) also bound ferric

siderophores with wild type affinities (Fig. 2, Table 2). Our Fhu$ construct was identical to that of Braun

et al (45). Regarding the β-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 G151. 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, Table 2). This reduction did not result from poor expression (Table 2), 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.

Interactions with colicins and bacteriophage. The $-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

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ligand selection. Likewise, the FhuA barrel recognized T5 and N80, and the transposition of the FepA N-

terminus into it did not change this specificity (Table 2). 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 Fhuβ to confer susceptibility to

colicin M. This discrepancy may stem from the low activity of our colicin M preparation.

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 $-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).

Uptake experiments with the mutant proteins, especially those containing the FhuA barrel, bore

relevance to these questions. In spite of its binding ability, the presence of the FepA N-terminus within it

made it unlikely for FepNFhu$ to transport Fc. Yet the chimera efficiently internalized the hydroxamate

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siderophore. Fc stimulated the growth of bacteria expressing FepNFhu$ as much as strains producing

FhuA (Fig. 3), 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 nM and 0.6 nM, respectively;

Fig. 2, Table 2), the chimeric protein displayed a comparable turnover number to the wild type protein (k3

values of 6.5/min and 4/min, respectively; Table 2). 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, Table 2).

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 (FepNFhuβ, FhuNFepβ, Fepβ and Fhuβ). 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 non-linear fit calculations from the Bound vs Total and Michaelis-

Menten equations (Figs. 2 and 3, insets) substantiated the accuracy of the measurements. For bacteria

expressing FepNFhuβ, FhuNFepβ and Fepβ, 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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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 non-specific permeability. The $-

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 4oC, 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.

As a further indication of the folding of 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-terminus (J. A. Coulton, personal

communication) 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

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domain. Like FepNFhu$ in ELISA, FhuNFep$ was indistinguishable from wild type FepA in flow

cytometry (Table 2).

Finally, neither chimeric protein conferred increased permeability to large antibiotics, whereas

bacteria expressing either Fep$ or Fhu$ showed enhanced susceptibility to bacitracin (1500 D; Table 2),

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 β-barrels.

Crosslinking with SulfoEGS. In the presence of formaldehyde, FepA crosslinks to another cell

envelope protein, TonB {for review see (4)}. However, skepticism about formaldehyde as an indicator of

proximity led us to test other crosslinking agents with more specific chemical targets. The bifunctional,

cleavable crosslinking agent SulfoEGS, selectively reacts with primary amines, and is too large (661 kD)

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 E. coli outer membranes, SulfoEGS

generated two prominent protein complexes that included FepA, of approximate molecular weights 100

(Band 1) and 120 kD (Band 2; Fig. 6). The identity of the crosslinked protein(s) in the 100 kD complex is

currently unknown. When cleaved, the 120kD 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 kD) 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 crosslinking

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

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Fep$, produced analogous results, reducing the levels of the 100 kD and 120 kD products to barely

detectable levels (Fig. 6).

Discussion

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$ 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

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globular domain, suggesting that the N-domain contributes to the overall efficacy and specificity of the

ligand binding process. However, the sub-nanomolar 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.

One of the mutant constructs (FhuNFepβ) 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:

k1 k3 FeXout + Receptor W FeX-Receptor 6 Receptor + FeXin . k2

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 .

Besides its high affinity, Fhu$ transported Fc in a TonB-dependent manner, with a better turnover

number than wild type FhuA (k3 in Table 2). 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 2, Fig. 3 (24,46,49,50)}. Therefore, even the least active

construct, FhuNFepβ, 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

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(see below), these data demonstrate that TonB does not function by an interaction with the N-terminal

150 amino acids of ferric siderophore receptors, in spite of indirect evidence from genetic suppression

(60-62), crosslinking (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 FhuNFepβ 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 non-

specific 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, that 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

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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, in spite of 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, 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 D, 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

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(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 crosslinking experiments reported herein provide

further contraindications to the static picture surmised from crystallography. When live bacteria were

exposed to sulfoEGS, FepA crosslinked to the major OM proteins OmpF/C and OmpA. Binding of

FeEnt eliminated the crosslinking, 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 crosslink to other

proteins. The failure of Fep$ to crosslink 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 crosslinking in the study of proposed

physical interactions between TonB and FepA, because of lack of control data on the crosslinking of TonB

to non-TonB-dependent OM proteins, uncertainties about the nature and identities of chemical targets of

the formaldehyde-activated residues within the crosslinked proteins, and the absence of quantitation of the

very small amount of total crosslinked products (4). The crosslinking 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-proteinA immunoblots} that were amenable to the identification of

all detectable crosslinked 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

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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 crosslinking 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 {Y272, F329; Y260 (49)} and an interior site {R316 (50) and

Y260 (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 suggests that the N-

domain exits the barrel as ligands traverse the OM. On this basis we propose that TonB directly or

indirectly promotes loop dynamics that force bound ligands into the underlying pore, but this 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 & chain” hypothesis (74). The possibility of hydrogen

bonds between the residues on the surfaces of the globular domain and the β-barrel walls suggests an

energetic barrier to this reaction mechanism, perhaps explaining the energy dependence of iron transport

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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, non-specific 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

The authors 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 This work was supported by NSF grant MCB9709418 and NIH grant GM53836.

Figure Legends

Figure 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 grey.

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Figure 2. FeEnt and Fc binding. The adsorption of 59FeEnt (open symbols) and 59FeFc (filled

symbols) was measured with metabolically inactive KDF541 expressing FepA or FhuA (F), FhuNFep$

(ªª), FepNFhu$ (««), Fep$ (G), and Fhu$ (�), carried on pHSG575.

Figure 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 those of KDF541. (Bottom) The transport of 59FeEnt (open symbols) and 59FeFc (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$ (*).

Figure 4. Expression, localization and non-denaturing electrophoresis of mutant proteins. Panel

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 ug of purified FepA, respectively; Lanes 4-10 each

contain lysates of 2. 5 x 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 2. Panel B: Localization. Inner and outer membranes from

bacteria expressing the chimeric proteins were fractionated on sucrose gradients, and 30 ug 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. Panel

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C: Non-denaturing SDS-PAGE. The left panel is a coomassie blue-stained LDS-PAGE gel; the right

panel is a western immunoblot 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, non-denatured FepA and FhuA,

respectively; lanes 6-8: OM fragments (30 ug) from KDF541 expressing FepA, FhuNFep$, and

FepNFhuβ.

Figure 5. Analysis of FepNFhu$$ by ELISA. Outer membranes from KDF541 carrying pITS23 (left) or

pFepNFhu$ (center), or purified, denatured FepA (right) 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, while the latter

two bind epitopes in surface loops of the barrel. Panels A and B plot mean values from 3 experiments;

Panel C shows the results of a single experiment.

Figure 6. Crosslinking of bacteria with Sulfo-EGS. A. Outer membranes from BN1071 (lanes 1-3)

or KDF541/pITS449 (lanes 4-6) were incubated with the cleavable crosslinker Sulfo-EGS, subjected to

SDS-PAGE, and stained with coomassie blue. Lanes 1 and 4: no crosslinking; lanes 2 and 5: crosslinked

by SulfoEGS. Lanes 3 and 6: crosslinked by SulfoEGS in the presence of 5 uM FeEnt. Lane 7:

molecular weight standards myosin (200 kD), $-galactosidase (116 kD), phosphorylase b (97 kD) bovine

serum albumin (66 kD), ovalbumin (45 kD), and carbonic anhydrase (31 kD). 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 kD and 36kD, 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 cell lysates of KDF541 harboring

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pITS23, (lanes 4-6) or pFep$ (Lanes 7-9) were either untreated (lanes 1,4, 7), or crosslinked with Sulfo-

EGS in the absence (lanes 2, 5, 8) or presence of 5 uM FeEnt (lanes 3, 6, 9), and cell lysates were

transferred to nitrocellulose and analyzed by immunoblot with anti-FepA MAb 45, developed with 125I-

proteinA.

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Genotype Reference Strains BN1071 F- thi, entA, pro, trp, rpsL (75) KDF541 BN1071 recA, fepA, fhuA, cir (24) KDF571 KDF541 tonB (24) AN193 F- pro leu, trp, thi, fhuA, lac, rpsL, entA (75) Plasmids pHSG575 (47) PITS449 fepA+ on pUC18 (24) pITS23 fepA+ on pHSG575 ¶ this study pFep$ fepA∆17-150 “ pFep$2 fepA∆3-150 “ pFepNFhuβ fepA1-152::fhuA160-723 “ pITS11 fhuA+ on pHSG575 “ pFhu$ fhuA∆5-160 “ pFhuNFepβ fhuA 1-155::fepA149-724 “ pFhuNFepβ2 fhuA 1-160::fepA153-724 “

Table 1 Strains and Plasmids.

¶ All constructions were analyzed as derivatives of the low copy plasmid PHSG575.

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Ferric Enterobactin Protein Ligands†

Binding¶ Transport§

MAbs‡

Antibiotics¢

Strain/plasmid Kd Cap Nutr¥ Km Vmax k3

ColB

ColD 33 45 N B E

Exp.^̂

KDF541 NB 0 0 NT 0 0 R R 0.15 0.13 14 0 0 0

“ /pFepA 0.4 64.0 18 0.40 411 6.4 105 4x104 7.8 14.1 14 0 8 1.2x105

“ /pFhuNFep$ 0.2 1.6 25* 1.7 1.6 0.9 103 4x101 7.6 8.3 14 0 8 1.5x104

“ /pFepA$ 0.6 4.3 20* 1.45 1.4 0.3 4x104 2x103 8.9 13.8 14 8 10 0.8x104

Ferrichrome ColM T5 NN80

AN193 NB 0 0 NT 0 0 R R R ND ND 14 0 8 0

“ /pFhuA 0.6 23 19 0.6 87 4 2x102 2x106 108 ND ND 14 0 8 ND

“ /pFepNFhu$ 0.6 1.6 19 2.5 9.8 6.5 R 105 107 0.2 0.18 14 0 8 9.4x104

“ /pFhu$ 1.0 3.0 21 3.6 25 8.3 2x102 105 4x107 ND ND 16 8 8 ND

Table 2. Biochemical properties of FepA and FepA mutants.

¶ Kd (nM) and Capacity (pMol bound /109 cells) were determined from the concentration dependence of

FeEnt binding, by analyzing the mean values from three independent experiments with Grafit 4

(Erithacus), using the “Bound vs Total” equation. For FeEnt, the mean standard errors for Kd and

Capacity were 16% and 4%, respectively; For Fc, the mean standard errors for Kd and Capacity were 15%

and 2% . NB: no binding.

¥ Nutrition tests express the diameters (mm) of growth halos. The results of a single experiment are

given, but the experiments were repeated several times without significant variation. An asterisk (*)

indicates that a very faint halo was observed (see Fig. 3).

§ Km (nM) and Vmax (pMol/min/109 cells) of uptake were determined from the concentration dependence

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of FeEnt transport, by analyzing the mean values from three independent experiments with Grafit 4

(Erithacus), using the “Enzyme Kinetics” equation. For FeEnt, the mean standard errors for Km and Vmax

were 17% and 4%, respectively. For Fc, the mean standard errors for Km and Vmax were 27% and 6%.

The turnover number for each protein (k3; molecules transported/min) was calculated by dividing Vmax by

capacity. NT: no transport.

† Colicin and bacteriophage susceptibility was determined by measuring the killing of KDF541

expressing the wild type and mutant proteins by limiting dilutions of colicins B, D, and M, and phage T5

and N80. The results are expressed as the titer of the killing agents on the different bacterial strains.

‡ Anti-FepA MAbs 33 and 45, that recognize sites in loops 2 and 4, respectively, were used to measure

the exposure of surface loops on the bacteria expressing the proteins of interest, relative to the FepA-

deficient strain KDF541. The tabulated values are mean fluorescent intensity. The data are from a single

experiment, but the experiment was repeated several times with little variation.

¢ The tabulated values are the diameter (mm) of the zone of clearing created by a 6 mm filter paper disk,

imbedded with an antibiotic (N: Neomycin, 30 ug/ml; E: Erythromycin, 15 ug/ml; B: Bacitracin, 10

IU/ml), on a lawn of the bacteria.

^ The expression of FepA and its chimeras (copies/cell) was measured by SDS-PAGE and immunoblots

with anti-FepA MAbs and 125I-protein A (Fig. 4).

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D.C. Scott et al., Fig. 2

[FeEnt] (nM)

0 2 4 6 8 10

pM

ol B

ou

nd

/109

Cel

ls

0

20

40

60

0 2 40

0.5

1

1.5

2

[Fc] (nM)

0 5 10 15 20

0

10

20

0 5 10 150

0.5

1

1.5

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D.C. Scott et al., Fig. 3b

[Fc] (nM)0 10 20 30 40

0

25

50

75

100

[FeEnt]( nM)0 2 4 6 8 10

pM

ol/m

in/1

0E9

cells

0

100

200

300

400

0 2 4 6 8 100

0.5

1

1.5

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Log Dilution1 2 3 4 5 6 7

A B C

Log Dilution

1 2 3 4 5 6 7

Ab

sorb

ance

0

0.5

1

1.5

2

2.5

Log Dilution1 2 3 4 5 6 7

D.C. Scott et al., Fig. 5

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Newton and Phillip E. KlebbaDaniel C. Scott, Zhenghua Cao, Zengbiao Qi, Matthew Bauler, John D. Igo, Salete M.C.

Exchangeability of N-termini in the ligand-gated porins of Escherichia coli

published online January 19, 2001J. Biol. Chem. 

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