doi:10.1016/j.jmb.2008.03.044 J. Mol. Biol. (2008) 378, 840–851

Available online at www.sciencedirect.com

Modulation by Substrates of the Interaction between theHasR Outer Membrane Receptor and Its SpecificTonB-like Protein, HasB

Julien Lefèvre1, Philippe Delepelaire2, Muriel Delepierre1

and Nadia Izadi-Pruneyre1⁎

1Département de BiologieStructurale et de Chimie, Unitéde Résonance MagnétiqueNucléaire des Biomolécules,Institut Pasteur, CNRS URA2185 28, rue du Dr. Roux,75724 Paris Cedex 15, France2Département de Microbiologie,Unité des MembranesBactériennes, Institut Pasteur,CNRS URA 2172, 75724 ParisCedex 15, France

Received 7 November 2007;received in revised form15 February 2008;accepted 21 March 2008Available online28 March 2008

*Corresponding author. E-mail addrAbbreviations used: ITC, isotherm

calorimetry; pmf, protonmotive forcdependent outer membrane transpo

0022-2836/$ - see front matter © 2008 E

TonB is a cytoplasmic membrane protein required for active transport ofvarious essential substrates such as heme and iron siderophores through theouter membrane receptors of Gram-negative bacteria. This protein spansthe periplasm, contacts outer membrane transporters by its C-terminaldomain, and transduces energy from the protonmotive force to thetransporters. The TonB box, a relatively conserved sequence localized onthe periplasmic side of the transporters, has been shown to directly contactTonB.While Serratia marcescens TonB functions with various transporters, HasB,

a TonB-like protein, is dedicated to the HasR transporter. HasR acquiresheme either freely or via an extracellular heme carrier, the hemophore HasA,that binds to HasR and delivers heme to the transporter. Here, we study theinteraction of HasR with a HasB C-terminal domain and compare it withthat obtained with a TonB C-terminal fragment. Analysis of the thermo-dynamic parameters reveals that the interaction mode of HasR with HasBdiffers from that with TonB, the difference explaining the functionalspecificity of HasB for HasR. We also demonstrate that the presence of thesubstrate on the extracellular face of the transporter modifies, via enthalpy–entropy compensation, the interaction with HasB on the periplasmic face.The transmitted signal depends on the nature of the substrate. While thepresence of heme on the transporter modifies only slightly the nature ofinteractions involved between HasR and HasB, hemophore binding on thetransporter dramatically changes the interactions and seems to locallystabilize some structural motifs. In both cases, the HasR TonB box is thetarget for those modifications.

© 2008 Elsevier Ltd. All rights reserved.

Edited by I. B. Holland

Keywords: HasB; TonB; heme transport; signal transduction

Introduction

The cell envelope of Gram-negative bacteriaconsists of the cytoplasmic membrane, the peri-plasm containing the peptidoglycan network andthe outer membrane. The outer membrane acts as aselective permeation barrier. Indeed, while mostabundant hydrophilic nutrients (b600 Da) needed

ess: nizadi@pasteur.fr.al titratione; TBDT, TonB-rter.

lsevier Ltd. All rights reserve

for bacterial growth diffuse passively into theperiplasm through outer membrane channels, raremetals and large organometallic cofactors such asiron siderophores and vitamin B12 must be activelytransported. This active transport through the outermembrane proceeds by coupling highly specificouter membrane transporters to an inner membranecomplex of proteins, ExbB, ExbD and TonB.1–3 TheTonB protein couples the inner membrane proto-nmotive force (pmf) to the outer membrane trans-porters. These TonB-dependent outer membranetransporters (TBDTs) share common structuralfeatures and are composed of two domains: aconserved N-terminal globular domain and a 22-stranded β-barrel inserted into the outer membrane.

d.

841Signal Transduction by an Outer Membrane Transporter

The N-terminal domain forms a “plug” that closesthe channel through the β-barrel.4–9 Some transpor-ters also contain a long N-terminal extension, thesignaling domain, that is involved in regulation oftranscription of their own gene.10

TonB is a three-domain protein containing an N-terminal transmembrane helix that anchors theprotein to the cytoplasmic membrane, a centralproline-rich domain that resides within the peri-plasm and a C-terminal globular domain thatdirectly contacts transporters in the outer mem-brane.11,12 There is currently no structure availablefor the entire TonB protein. However, three struc-tures have been reported for the TonB C-terminaldomain. The oligomeric state of TonB fragments isvariable and related with the length of the recombi-nant construct. Fragments longer than the last 96residues are monomeric, whereas smaller fragmentsare dimeric.13,14 TonB dimerization has also beenobserved in vivo, at least while associated with theExbB/ExbD proteins.15,16 The relevance of thisdimerization for TonB activity is unclear. Twostructures of the C-terminal domain of TonB,complexed either with the vitamin B12 transporter,BtuB, or with the hydroxamate siderophore ferri-chrome transporter, FhuA, are now available in thepresence of their respective substrates. The stoichio-metry of the complex is 1:1. The structures show thatTonB interacts with one part of the periplasm-exposed surface area of the transporter. A criticalregion for this interaction involves a conserved N-terminal region called the TonB box. In the complex,the TonB box displaces TonB's fourth β-strand toform an interprotein β-sheet.17,18 While providing adetailed view of the interaction between TonB andTBDTs in a complex, structures alone do not explainhow this interaction leads to substrate transport. It iscurrently accepted that the plug undergoes largestructural changes to allow ligand entry into theperiplasm. Under TonB action, the plug mightundergo conformational changes within the barrelto form a transient channel or be pulled out of thebarrel, either as a globular unit or in an unfoldedstate.19 Currently available experimental data areconsistent with those models.20–22

In many species, there is only one TonB proteinshared by the various TBDTs produced by the species.For instance,Escherichia colihas only one TonBproteinand seven knownTBDTs that all compete for a limitedamount of TonB.23 Some bacterial species containseveral genes encoding TonB homologs in theirgenome. Some of them exhibit partially redundantfunctions with distinct specificities.24 In addition tothe TonB protein, the Gram-negative bacterium S.marcescenspossesses a TonB-like protein namedHasB,which is a component of the heme acquisition Hassystem.25 This system, found in several Gram-negative bacteria, internalizes heme for iron utiliza-tion; heme being amajor iron source for pathogenic orcommensal bacteria in mammals. The Has systeminvolves an extracellular protein (the HasA hemo-phore) that captures free or hemoprotein-boundheme and delivers it to a specific outer membrane

transporter (HasR).26 HasR binds free heme with asubmicromolar affinity. The system is more efficientwith the hemophore due to the very high affinity ofHasA for heme (Ka=5×10

10 M−1) and the strongaffinity of HasA for its cognate transporter HasR(KaN10

9 M−1).27,28 While the interaction of thetransporter HasR with heme or hemophore andheme transfer from the hemophore to the transpor-ter do not require energy, the ensuing steps of hemeinternalization through the transporter and ejectionof the empty hemophore from the transporterrequire energy provided by TonB or HasB.29,30HasB shares 17% identity with E. coli TonB and

26% identity with S. marcescens TonB. The analysis ofits sequence reveals the same three-domain struc-tural organization as that of TonB. In S. marcescens,both HasB and TonB are active in heme uptake viathe HasR transporter, but HasB cannot replace TonBfor the other TonB-dependent processes. Reconstitu-tion of the S. marcescens hemophore-dependentsystem in E. coli demonstrated that HasB cannotreplace TonB for any of its functions. However, amutation in the HasB transmembrane segmentenables the protein to be active for heme uptakevia HasR expressed in E. coli in a tonB− background,without complementing the other TonB-dependentfunctions such as ferric siderophore internalization.Although this mutation does not change HasBspecificity, it renders it functional in E. coli, likelyvia better adaptation of the HasB protein to the E. coliExbBD complex.25

There is a great deal of evidence that HasBbehaves as a specific TonB-like protein only in-volved in heme uptake through the HasR transpor-ter. In order to determine the basis of this specificityand to understand how HasB functions, we studiedthe in vitro interaction of HasR with HasB133, aperiplasmic domain of HasB (residues 133–263), andcompared it with that of an equivalent domain ofTonB. The Has system had been completely recon-stituted in E. coli and all in vivo data related tofunctioning of HasR transporter have been obtainedin E. coli. This is why a periplasmic fragment of E.coli TonB was used for the comparative interactionstudy. Two other TBDTs have also been tested withregard to their interaction with HasB. One of themwas FhuA (ferrichrome transporter) of E. coli, forwhich the interaction with TonB has been exten-sively studied.31,32 The other was HemR of S.marcescens. This TonB-dependent heme transporterbelonging to the Hem system represents anotherheme acquisition system of S. marcescens. HasB doesnot function with HemR33 and does not allowferrichrome internalization.25 In the present com-parative study, we show that HasB is highly specificfor the HasR transporter.To gain more insight into the mechanism of

active transport by specific transporters, westudied the interaction of HasB133 with HasRloaded with its various ligands—heme, apo orholoHasA. We show that fixation of the ligand onthe transporter induces a signal that is transmittedfrom the extracellular face to the periplasmic part of

Fig. 2. ITC analysis of the interaction of HasB133 withapoHasR (a) and TonB116 with apoHasR (b). Representa-tive experiments are shown. In each case, the heat signal isshown (top) together with the binding isotherm derivedfrom this signal (bottom).

842 Signal Transduction by an Outer Membrane Transporter

the transporter involved in the interaction withHasB. The transmitted signal depends on the natureof the ligand.

Results

HasB133 is monomeric

TonB periplasmic fragments have been observedas monomeric, dimeric and heterogeneous mono-meric–dimeric forms in solution. In order todetermine the oligomeric state of our HasB peri-plasmic fragment, we analyzed the molecularweight distribution of HasB133 in sedimentation/diffusion equilibrium experiments. Satisfactory fitsto the observed distributions were obtained using aone-solute ideal solution model with a molecule ofmolecular mass of 14,350 (±100) Da (Fig. 1). Thesevalues are in agreement with that of the calculatedmolecular mass of 14,405 Da, with the variationsremaining within the limits of the method. Thisresult clearly indicates that purified HasB133 at aconcentration of at least 1×10−4 M is monomeric,since no trace of multimers was detected.

HasB133 interacts with HasR with a 1:1stoichiometry and high affinity

In order to test the capacity of HasB133 interactionwith the HasR transporter and to characterize thisinteraction, we analyzed it by microcalorimetry,isothermal titration calorimetry (ITC). ITC titrationof apoHasR with HasB133 gave a very strong ne-gative enthalpic signal (Fig. 2a). The data fit to asingle-site model with a 1:1 stoichiometry, an affinityconstant of 7.5×107 M−1 (Kd=13 nM) and a ΔH of−80.9 (±1) kJ mol−1. The interaction between HasRand HasB133 is enthalpically driven, indicating thatseveral polar processes are involved in this interac-

Fig. 1. Sedimentation/diffusion equilibrium ofHasB133 (3×10−5 M). Absorbance at 277 nm was scannedas a function of the distance to the rotation axis. Bottom,experimental points (open circles) were used to generate afitted curve (continuous line) using as a model an idealsolutionwith one solutemolecule. Top, residuals in termsofstandard deviations from the weighted fit.

tion, consistent with the crystal structure of TBDT–TonB complexes in which several hydrogen bondsand one electrostatic interaction are involved incomplex formation. The entropy contributes unfa-vorably to the free energy of binding (TΔS=−36 (±1)kJmol−1). This unfavorable entropy contribution canbe associated with conformational changes of resi-dues in the complex.

Characterization of the HasR–HasB133interaction

In vivo studies have shown that both HasB andTonB are functional with HasR.25 In order todetermine the exclusivity of the high-affinity inter-action between HasR and HasB, we studied the invitro interaction of HasR with TonB116, a TonBperiplasmic fragment of a size comparable to thatof HasB133. It is noteworthy that, as reported byKoedding et al., TonB116 is also monomeric.34 ITCtitration of HasR with TonB116 gave an enthalpicsignal five times lower than that observed for theHasR–HasB133 interaction (Fig. 2b). Fitting of thedata by a single-site model indicates a 1:1

843Signal Transduction by an Outer Membrane Transporter

stoichiometry and an affinity constant of2.2×106 M−1 (Kd=4.5×10

−7 M), 34 times lowerthan that measured for the interaction withHasB133. The HasR–TonB116 interaction isenthalpy–entropy driven (ΔH=−16.3 (±2) kJ mol−1,TΔS=19.8 (±2) kJ mol− 1). Consequently, HasRinteracts differently with TonB and HasB (Table1). While TonB enables substrate transport viavarious transporters, HasB functions only withHasR. What is the behavior of HasB with theother TBDTs? Is the high affinity of HasB forHasR exclusive of this pair of proteins?FhuA binds to HasB133 with an affinity constant of

2.6×105M−1 (Kd=3.8×10−6M) to forma1:1 complex.

The affinity constant and the thermodynamic para-meters of FhuA–HasB133 interaction are verydifferentfrom those obtained for HasR–HasB133. This lowaffinity between FhuA and HasB133 could explainwhy HasB is not active for ferrichrome uptake.27ITC titration of HemR of S. marcescens with

HasB133 did not show any detectable enthalpicsignal higher than that corresponding to the heat ofdilution. The highest HemR concentration used forthis titration was 9 μM. A titration within the samerange of concentration (5 μM) of HemR withTonB116 showed an enthalpic binding signal.HemR binds to TonB116 with an affinity constantof 3.9×106 M−1 (Kd=2.6×10

−7 M). The HemR–TonB116 interaction is enthalpy–entropy driven. Thefact that no enthalpic signal is detected betweenHemR and HasB133 indicates that either HasB133interacts only via hydrophobic contacts or, morelikely, that in our experimental conditions, HasB133does not interact with HemR. In the latter case, wecan conclude that the putative affinity constantwould be lower than 105 M−1, a value much lower(at least 750-fold) than that obtained for the HasR–HasB133 interaction.Nonspecific interactions of TonB with proteins or

peptides have been already observed by cross-linking and phage panning.35 The ITC titration ofthe membrane preparation, which did not containHasR, with either HasB133 or TonB116 did not showany enthalpic signal (data not shown). Although wecannot exclude an unspecific interaction, its con-tribution in our enthalpic signal is negligible.The thermodynamic parameters of all HasB133/

TonB116–transporter interactions are summarized inTable 1.

Table 1. Stoichiometries (N), affinity constants (Ka),ΔH and TΔTBDTs at 25 °C

Binding partners N Ka (M−

HasB133+apoHasR 1.03±0.05 7.5 (±0.2)HasB133+FhuA 1.13/0.96 1.9×105/3.HasB133+HemR — —TonB116+apoHasR 1.01±0.08 2.2 (±0.1)TonB116+HemR 0.99±0.05 3.9 (±1.4)

Values are the averages of three experiments±standard error of the mtwo sets of experimental values are indicated.

Contribution of the TonB box to the HasR–HasBinteraction

Like other TBDTs, HasR contains a conservedmotif referred to as the TonB box, composed ofDSLTVLGA. The TonB box has been shown to benecessary for recruiting TonB and largely participatesin the interaction with the transporter in the alreadyknown complex structures. In order to determine thecontribution of the HasR TonB box in the interactionwithHasB133, we examined the behavior of amutatedtransporter in which the TonB box motif was deleted(HasRΔtbb). The mutated transporter was correctlylocalized at the outer membrane and was purifiedusing the same protocol as for the wild-typetransporter. However, HasRΔtbb was inactive in invivo heme acquisition (data not shown). Titration ofHasRΔtbb with HasB133 displayed an enthalpicsignal. This observation indicates that, unexpectedly,HasRΔtbb is still able to interact with HasB133. Thedata fit to a single-site modelwith a 1:1 stoichiometry,an affinity constant of 3×106 M−1 (Kd=3.3×10

−7 M)and a ΔH of −31.4 (±0.5) kJ mol−1. The thermody-namic parameters of this interaction reveal thatbinding of HasRΔtbb with HasB133 remainsenthalpy-driven in the absence of the TonB box. Thetitration of this mutant with TonB116 up to aconcentration of 1.2 10−4 M did not show any inter-action enthalpic signal.Binding of HasRΔtbb to HasB133 shows that

besides the TonB box, one or several other regionsof HasR are involved in the interaction with HasB.With TonB116, the enthalpic contribution of interac-tion of remaining regions, if any, was not enough tobe detected by ITC in our conditions.

The HasR–HasB interaction is modified by thepresence of substrate

The HasR transporter recognizes heme andhemophore (both apo and holo forms) on itsextracellular side, while it binds HasB on itsperiplasmic side. In order to determine whetherbinding of substrates modifies the interaction withHasB133, we analyzed the interaction of HasB133with HasR previously loaded with its variousligands. For this, complexes between apoHasR andeither heme, apo or holohemophore were formedand used for titration experiments with HasB133. The

S of binding of HasB133/TonB116 with apoHasR and other

1) ΔH (kJ mol−1) TΔS (kJ mol−1)

×107 −80.9±1 −36±1.03×105 −9.4/−12.1 20.74/19

None —×106 −16.3±2.0 19.9±2.0×106 −35.0±2.6 2.53±2.0

ean. For the second line, two experiments have been done and the

Table 2. Thermodynamic parameters for binding of HasB133 and TonB116 with various forms of wild-type or mutatedHasR in 20 mM sodium phosphate buffer, pH 7, and 0.08% ZW 3-14 at 25 °C

Binding partners N Ka (M−1) ΔH (kJ mol−1) TΔS (kJ mol−1) ΔG (kJ mol−1)

HasB133+apoHasR 1.03±0.05 7.5 (±0.2)×107 −80.9±1 −36±1.0 −44.9±0.2HasB133+holoHasR 0.99±0.01 7.0 (±1)×107 −77.4±0.7 −32.6±0.8 −44.8±0.3HasB133+(apoHasA+apoHasR) 0.98±0.01 4.76 (±0.8)×107 −117.0±0.9 −73.2±1.1 −43.8±0.4HasB133+(holoHasA+apoHasR) 1.1±0.01 4.73 (±0.6)×107 −107.7±0.9 −63.9±0.9 −43.8±0.3TonB116+apoHasR 1.01±0.08 2.2 (±0.1)×106 −16.3±2.0 19.9±2 −36.2±0.1TonB116+holoHasR 1.06±0.04 2.4 (±0.4)×106 −12.0±2 24.4±2 −36.4±0.3TonB116+(apoHasA+apoHasR) 1.04±0.04 2.8 (±0.5)×106 −45.2±0.5 −8.4±0.7 −36.8±0.4TonB116+(holoHasA+apoHasR) 1.1±0.01 3.0 (±0.5)×106 −41.7±0.6 −4.72±0.7 −37.0±0.35HasB133+(apoHasRΔtbb) 0.99±0.01 3.02 (±0.4)×106 −31.4±0.5 5.6±0.6 −37.0±0.3HasB133+(holoHasA+apoHasRΔtbb) 1.1±0.01 3.5 (±0.8)×106 −31.1±0.7 6.2±0.9 −37.3±0.5

Fig. 3. Thermodynamic profiles for binding of HasB133with various forms of HasR in 20 mM sodium phosphatebuffer, pH 7, and 0.08% ZW 3-14. ΔG is in black, ΔH is ingray and TΔS is in white. AA, apoHasA; hA, holoHasA;aR, apoHasR. HoloHasR is the heme-loaded form ofHasR.

844 Signal Transduction by an Outer Membrane Transporter

heme-loaded form of HasR will hereinafter bereferred to as holoHasR.

Heme

The presence of heme slightly modifies thermo-dynamic parameters without changing the affinityconstant value of the HasR–HasB133 interaction(Table 2). The ΔH value, representing polar interac-tions, is less negative and therefore contributes lessto the affinity, whereas the TΔS contributiontowards the affinity of holoHasR–HasB133 complexfavors more compared with apoHasR. These varia-tions indicate that when HasR is loaded with heme,weaker or lesser polar interactions are involved incomplex formation compared with the apo-trans-porter. Since the affinity constant remains un-changed, this decrease in polar interactioncontribution is compensated for by an increase inentropy contribution. Such observations are referredto as “enthalpy–entropy compensation,” producingonly small changes in ΔG and thus in the affinityconstant.

Hemophore

The presence of apoHasA strongly modifies thethermodynamic parameters of the interactionbetween the transporter and HasB133 without sig-nificant change in Ka (below twofold) (Table 2). In thesame manner as mentioned above for heme, thechange in TΔS is compensated for by theΔH change,keeping ΔG and the affinity constant almostunchanged (Fig. 3). However, the magnitude andsigns of these changes are different from those thatoccur upon heme binding. While for apoHasA bin-ding, ΔΔH=ΔH[(substrate+apoHasR)+HasB133]−ΔH(apoHasR+HasB133) is −36 kJ mol−1, only achange of 3.4 kJ mol−1 is induced by heme binding.In the presence of apoHasA, theΔH of the interactionbetween HasR and HasB133 becomes increasinglynegative, indicating that more or stronger polarinteractions are involved in complex formation and/or induced by it. This additional contribution of polarinteractions compensate for the decrease in TΔS(ΔTΔS=−37.2 kJ mol−1).The binding of holoHasA to the transporter

induces the same effect of enthalpy–entropy com-

pensation (Fig. 3). Both HasA apo and holoformssimilarly modify the thermodynamic parametersof the HasR–HasB133 interaction: ΔH and TΔS be-come more negative. However, a slight difference isobserved between the two forms of HasA. In thecase of the complex (holoHasA+apoHasR) +HasB133, enthalpy and entropy terms are slightlydifferent (less negative) from those obtained with(apoHasA+apoHasR)+HasB133. These differencesare of the same magnitude and have the same signsas those observed when only heme binds to thetransporter. As already shown in our previousstudy, upon protein–protein interaction betweenholoHasA and apoHasR, heme is transferred toHasR.29 Therefore, the binding of heme to HasR,either directly or when transferred from HasA,induces a similar influence on HasB133 binding.Hence, the binding of holoHasA to the transporterresults from the additive effect of apoHasA andheme binding.

The presence of HasR substrates is alsosignaled to TonB

The presence of heme on HasR by enthalpy–entropy compensation induces a decrease in polar

845Signal Transduction by an Outer Membrane Transporter

processes and an increase in the entropy contribu-tion in the HasR–TonB116 interaction (ΔΔH=4 kJmol−1, Δ(TΔS)=4.6 kJ mol−1). These changes are ofcomparable magnitude and have the same signs asthose observed above for HasB133 by heme bindingto HasR. ΔG and thus the Ka values do not vary.Similarly to the HasR–HasB133 interaction, when

apo or holoHasA is present on the transporter, ΔHof the HasR–TonB116 interaction becomes morenegative, indicating that more polar interactionsare involved. This change compensates for theentropy change maintaining thus ΔG and Ka valuesunchanged. The presence of heme, apohemophoreor both on the transporter seems to produce thesame effect, i.e., comparable magnitude and thesame sign, for the interaction of HasR with eitherHasB133 or TonB116.

The substrate presence on the transporter is notsignaled to HasB in the absence of the TonB box

Numerous studies have shown that the TonB boxof TBDTs undergoes conformational rearrangementswhen the ligand binds to the transporter. This motifwas presumed to indicate the occupation state of thetransporter to the TonB protein. Given that the HasRmutant lacking its TonB box still interacts withHasB133 and that the HasR–HasB133 interaction isinfluenced by the presence of heme and hemophore,a logical question arises: Is the TonB box the target ofthe signal and/or is it involved in the signaltransduction process? To address this question, westudied the interaction of either a preformed complex(holoHasA+HasRΔtbb) or holoHasRΔtbb andHasB133 by ITC. Prior to this experiment, theinteraction of HasRΔtbb with hemophore (apo/holo) was analyzed. ITC titrations were identical tothose obtained with the wild-type transporter,showing that the mutant binds the two forms ofHasA in the same manner as the wild-type transpor-ter. Moreover, the UV–visible spectra of a 1:1complex between the holohemophore andHasRΔtbbwas identical to that obtained with wild-type HasR,indicating that heme was transferred from thehemophore to HasRΔtbb (data not shown).The fitted titration data of either the preformed

complex (holoHasA+HasRΔtbb) or holoHasRΔtbbwithHasB133 does not show substantialmodificationof thermodynamic parameters compared with thoseobtained for HasRΔtbb with HasB133. As shown inTable 2, ΔH[(holoHasA+HasRΔtbb)+HasB133]=ΔH(HasRΔtbb +HasB133), idem for ΔH(holo-HasRΔtbb+HasB133). Therefore, the interaction ofthe HasRΔtbb mutant with HasB133 is not modifiedby the presence of either heme or hemophore or both.

Discussion

TonB is a key protein in active transport of essentialsubstrates through the outer membrane transportersof Gram-negative bacteria. It spans the periplasm,contacts cognate outer membrane transporters via its

C-terminal domain and transduces energy from thecytoplasmic membrane pmf to the transporter,enabling substrate internalization. Some bacterialspecies contain several genes encoding TonB homo-logs in their genome. HasB is such a TonB-likeprotein belonging to the Has system from S.marcescens. It has been shown to be specific to HasRbut not to heme transporters.25,33 In order to under-stand how a specific TonB-like protein functions, weexpressed a C-terminal periplasmic fragment ofHasB and studied its interaction with the HasRtransporter. This study concerns the first step ofpartner recognition occurring in the absence ofenergy.Several studies have shown that the oligomeric

state of TonB is variable and related to the length ofthe recombinant constructs. Indeed, C-terminalfragments encompassing the last 96 residues aremonomeric and smaller fragments are dimeric insolution.34 In the case of the HasB protein, amonomeric form was expected for HasB133. Analy-tical ultracentrifugation reveals that HasB133 isindeed monomeric. Furthermore, its oligomericstate does not change upon HasR interaction, as a1:1 stoichiometry complex is obtained.

HasR is the specific substrate of HasB133

The first indication of HasB specificity for HasR isthe high value of the affinity constant (Kd=13 nM)compared with that reported in the literature for theperiplasmic fragment of TonB with other TBDTs.Indeed, apparent dissociation constants in themicromolar range have been determined by invitro studies between various TonB fragments andsome siderophore transporters.31,36 A lower appar-ent Kd in the nanomolar range has also beenreported for the FhuA–TonB complex, but with a1:2 stoichiometry. In this case, it has been postulatedthat FhuA first binds a TonB molecule with reducedaffinity (Kd in the micromolar range), followed by arearrangement of this initial complex and thenbinding of a second TonB molecule with a Kd inthe nanomolar range. Such a rearrangement has notbeen evidenced in other in vitro interaction studies ofTonB–TBDT. In our case, HasR titration withHasB133 up to a molar ratio of 1:3 excludes theexistence of such a second binding site. Comparisonof affinity constant values obtained for TonB116 orHasB133 interacting with HasR and other TBDTsconfirms that HasR is a specific substrate of HasB133.Indeed, affinity of HasB133 for HasR is about 34times stronger than affinity of TonB116 for HasR orHemR. The high affinity of HasB133 for HasR is notobserved for the other tested TBDTs, FhuA andHemR, for which the affinity of HasB133 is at least280 times lower. Hence, the higher affinity ofHasB133 for HasR is specific to the couple HasB/HasR, and is not due to the HasB mode of binding tothe transporter, whatever the transporter.Comparative analysis of the thermodynamic

parameters of interactions provides complementaryexplanations for the specificity of HasB for HasR. As

846 Signal Transduction by an Outer Membrane Transporter

determined by our ITC experiments, the HasR–HasB133 interaction is enthalpy-driven, demonstrat-ing that polar processes are dominant in theinteraction. The negative sign (unfavorable) of theentropic term usually seems to indicate that the lossof degrees of freedom has a greater effect than thegain in hydrophobic contacts. The unfavorableentropy could also be contributed by ordering ofwater molecules in and around the protein/proteininterface upon complex formation. The entropic costobserved in HasR–HasB133 complex formation canbe an indication of the disorder-to-order transitionof one or several regions of HasR and/or HasB. Itwould thus appear that these regions becomestabilized and/or structured upon HasR–HasB133interactions. In the case of the other transporterstested with HasB133 and with TonB116, includingTonB116–HasR interaction, both enthalpy andentropy terms are favorable. This suggests that inthese interactions, the gain in hydrophobic contactshas greater effect, either by the presence of morehydrophobic contacts or by lesser contribution ofconformational entropy compared with HasR–HasB133. Whatever the origin of these thermody-namic parameter differences, the binding mode ofHasR–HasB133 interaction is different from theothers tested in our study. This difference couldexplain the functional specificity of HasB for HasRpreviously observed in vivo.25

Signal of substrate presence

The HasR transporter binds heme, apo andholohemophore on its extracellular face and HasBon its periplasmic side. In this work, we show thatthe presence of the substrate (heme or apo/holoHasA) is not required for the HasR–HasB133interaction. These properties raise the question ofthe influence of the presence of the substrate on theHasR transporter in this interaction.Surface plasmon resonance and in vivo cross-

linking experiments showed that the presence of aspecific siderophore on the transporter enhanced theaffinity or the amounts of the TonB–transportercomplex.31,32 However, other studies, also doneusing surface plasmon resonance, revealed nodifference in the apparent affinity of the transporter–TonB complex when the substrate was present onthe transporter.36,37 Nevertheless, it cannot beexcluded that the influence of substrate bindingupon the transporter, if any, may be undetectable bythis method or is dependent upon the transporterand/or the substrate.In this work, we show that the presence of the

substrate on the extracellular face of the HasRtransporter changes the thermodynamic parametersof the interaction between HasR and HasB133 on theperiplasmic face. Although binding of each of thesubstrates has the same enthalpy–entropy compen-sation effect, maintaining the affinity of HasR–HasB133 unchanged, the signal for heme presencedoes not create the same effect as that given uponhemophore binding. When HasR is loaded with

heme, the nature or the number of polar interactionsbetween HasR and HasB133 is slightly decreasedcompared with the unloaded form. Conversely, thepresence of hemophore on the transporter istransduced by a gain in polar interactions uponHasR–HasB133 complex formation. Concomitantly,the entropic term becomes more negative (moreunfavorable) in the presence of hemophore. Thehigh negative value of ΔS can be associated withconformational restriction of one or several regionsof HasR and/or HasB133. There is an entropic costassociated with the disorder-to-order transitionalready observed in the HasR–HasB133 interaction,but which is accentuated by the presence ofhemophore on HasR. In the latter case, it iscompensated for by more polar processes. Thisenthalpy–entropy compensation has been reportedin several examples of functional intrinsically dis-ordered protein domains involved in cellular signaltransduction, regulation of transcription, etc.38 Allthese results seem to indicate that one or severalregions involved in HasR–HasB133 interactionbecome more stabilized or structured by supple-mentary polar processes in the presence of apo-HasA. The N-terminal signaling domain of HasRcould be one of these regions.When holoHasA is the substrate, its binding

produces an additive effect of apoHasA bindingplus that of heme binding to HasR. This result isconsistent with the fact that the concomitantpresence of heme and apoHasA (or only holoHasA)on the HasR transporter is the signal activating thecascade of transcription regulation of the has operonencoding for proteins involved in the Has systemsuch as HasR, HasA and HasB.39 The binding ofeach of these substrates alone (heme or hemophore)cannot activate the signaling cascade transmittedthrough HasR and its N-terminal signaling domainup to the anti-sigma–sigma factors involved in hasregulation transcription.40,41 Together, this findingand our results show that, although the HasAhemophore is not transported by HasR, its presenceis a signal that is transferred up to the periplasmicface of HasR. Heme is the transported substrate, andits presence on the HasR extracellular face should bedetected by HasB required for its internalization.The hemophore is not transported, but its presencemust also induce a signal to HasB that gives therequired energy for its ejection.These effects induced by substrate binding are not

specific to the HasR–HasB133 interaction, since theyare also observed with TonB116. Similar networks ofinteraction between HasR and TonB116/HasB133 arethus involved in signal transduction by the substratepresence on HasR.

Role of the TonB box

Several studies have shown that the TonB box isrequired in transport and signal transduction byTBDTs. The fact that the HasR mutant lacking itsTonB box still interacts with HasB133 indicates thatone or several other regions besides the TonB box are

847Signal Transduction by an Outer Membrane Transporter

involved in the interaction with HasR. In a recentstudy of TonB/FepA interaction, it has also beensuggested that TonB could bind at a different siteprior to interaction with the TonB box.42 In HasR–HasB113 complex, this additional site is not influ-enced by the presence of heme and HasA, sincethermodynamic parameters remain identical. Con-sequently, assuming that no structural changeaccompanies deletion of the TonB box, the signalinduced by the presence of substrate needs the TonBbox in order to be transmitted.The TonB-box in TBDTs for which the structure is

available has been observed to be either ordered,although devoid of regular secondary structures, ordisordered. In the transporter–TonB complex struc-ture, the TonB box is recruited to form a parallel β-strand with the three-stranded β-sheet of TonB. Ourthermodynamic parameters are consistent with thefact that the HasR TonB box becomes structuredupon interaction with HasB133. The loss of 50 kJmol− 1 of the ΔH value of HasRΔtbb+HasB133interaction compared with HasR+HasB133 is alsoconsistent with the loss of several hydrogen bondsthat could have been involved in a β-strandformation of the TonB box with HasB.All HasR extracellular substrates, free heme,

apoHasA and holoHasA, differentially modify theinteraction with HasB. However, in all cases, thosemodifications are transmitted via theHasRTonB box.While the presence of heme in the extracellular faceof the transportermodifies the nature of the involvedinteractions between HasR and HasB133/TonB116,conformational changes occurring by hemophorebinding to the transporter seem to locally stabilize astructural motif implicating the TonB box. A recentstructural study of FpvA supports this finding. Inthis study, the location of the N-terminal signalingdomain and the TonB box conformation weremodified upon binding of the substrate on theextracellular face of the transporter.43 Comparablestructural modifications of HasR are consistent withour themodynamic analysis.

Bioinformatic analysis identified 144 Gram-nega-tive bacteria possessing an annotated tonB gene.Among these, 60 bacteria have more than oneannotated tonB gene. Some organisms such asPseudomonas syringae possess up to nine tonBsequences, and several are linked to putativetransporter genes.44 In this study of such a specificTonB-like protein, we reveal that HasB interactswith HasR in a way clearly distinct from TonB. Theexistence of such a specific TonB-like protein mightbe a mechanism for overcoming the potentialdeficiency of TonB and dedicating a “specific energytransmitter” to a vital system. The study of theseTonB homologues will shed light on the first step ofpartners recognition involved in “TonB-dependent ”processes. Our interaction study between threeproteins is well suited for deciphering the signaltransduction mechanism coming from the extracel-lular environment and propagated through amembrane transporter to the periplasm. Furtherstructural information on HasB alone and com-plexed with various forms of transporter HasR willbe necessary to better understand this mechanism.

Materials and Methods

Strains and plasmids

The HasB133 domain was defined by its sequencehomology with TonB using as template the monomericTonB periplasmic fragment already studied.45 This domaincomprises the last 131 residues of HasB (residues 133–263)plus an additional N-terminal methionine (Fig. 4). TheHasB133 gene was amplified by PCR from plasmidpHasBpuc.46 The reaction mixture included proofreadingDNApolymerase Pfu (Stratagene) and each primer thatwedesigned to incorporate 5′ EcoRI and 3′ HindIII sites forcloning the amplified product into the plasmid pBAD24.The sequence of the PCR forward primer was as follows:5′-CAGGAGGAATTCACCATGAAGGTTCAGGAG-CAAAGCGT-3′. The EcoRI site is underlined. The

Fig. 4. Clustal W alignment[http://www.ebi.ac.uk/clustalw/]of HasB (top) and E. coli TonB(bottom) sequences. Asterisks indi-cate conserved residues in twoproteins. Putative transmembrane(shaded) and proline-rich regionsare depicted.

848 Signal Transduction by an Outer Membrane Transporter

sequence of the PCR reverse primer was as follows: 5′-GCGTGGAAGCTTGCGTCAAAAACCCGCGC-CGCGCAAACTGAAATC-3′. The HindIII site isunderlined; the sequence in boldface type corresponds tothe hasB sequence. PCRproductswere digestedwith EcoRIandHindIII and ligated to the expression plasmid pBAD24that had been digested by the same enzymes. The plasmidsequence was verified on a DNA sequencer by GenomeExpress Company.Plasmid pFR2 encodes wild-type HasR under arabinose

control.29 Plasmid pFR2tbb encodes HasRΔtbb, a HasRmutant lacking the TonB box. It was generated using twomutagenic oligos, deltatbb1(5′-GGCGCGCTGGCGTT-GGGCGGCAACAACGCC-3′) and deltatbb2 (5′-GGCGT-TGTTGCCGCCCAACGCCAGCGCGCC-3′) as primers ina PCR reaction with original plasmid pFR2 as a template.The mutation was verified by sequencing and reintro-duced in an otherwise wild-type pFR2 by exchanging the0.7-kb EcoRI–NcoI fragment carrying the mutation. TheHasRΔtbb thus carries a deletion of eight residues,DSLTVLGA (100–107).Plasmid pTB116, a pET30a derivative kindly provided

by W. Welte, encodes TonB116, a C-terminal fragmentcontaining residues 124–239 of TonB.34

HemRhis, a C-terminal His-tagged HemR, was over-expressed in BL15 cells, under IPTG control, containing apET22 derivative plasmid. This pHemRhis plasmid waskindly provided by Najla Benevides-Matos.

Protein expression

HasB133 was expressed as recently reported.46 Wild-type and mutant HasR,29 HasA47 and TonB116

14 wereexpressed as previously described. HemRhis wasexpressed in LB medium at 30 °C. When cells reached anoptical density of 0.9 at 600 nm, IPTG (2 mM) was addedand the culture was incubated for 2 h. To simplify,HemRHis will hereinafter be referred to as HemR.When necessary, kanamycin, spectinomycin and chlor-

amphenicol were added at 50 μg/ml each. Ampicillin wasadded at a concentration of 100 μg/ml.

Protein purification and biochemical techniques

Two chromatography steps, cation exchange and gelfiltration, were used for HasB133 purification as recentlydescribed.46

HasAwas purified as previously described.48 HasR andHasRΔtbb were obtained by membrane preparation asearlier reported.29 The holoforms (heme-loaded) of HasAand HasR were obtained by addition of 1.2-fold molarexcess of freshly prepared hemin solution to theapoprotein.48 Heme excess was eliminated by gel filtra-tion. Complexes between apoHasA/holoHasA and HasRused for ITC experiments were formed by adding anexcess of ligand to purified HasR sample and afterremoval of excess by filtration using a membrane with amolecular mass cutoff of 100 kDa. It is noteworthy that inour previous studies, we showed that the HasA–HasRcomplex was stable and irreversible in vitro, consistentwith the fact that ejection of HasA requires energy.29,30

HemR was purified after membrane solubilization withZW3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-pro-pane sulfonate; Calbiochem). Membrane solubilizationwas performed using the same protocol as described forHasR. HemR was purified by affinity chromatographywith a 5-ml Hi-Trap chelating HP column (GE HealthcareLife Science) charged with Ni2+ ions and equilibrated with

buffer A (20 mM sodium phosphate, pH 7.4, 0.5 M NaCl,0.08% ZW 3-14, 5 mM imidazole). The bound proteinswere then eluted with 20 column volumes of a lineargradient of buffer A to 100% of buffer B (20 mM sodiumphosphate, pH 7.4, 0.5 M NaCl, 0.08% ZW 3-14, 0.5 Mimidazole) at a flow rate of 5 ml/min. A second step ofsize-exclusion chromatography of fractions containingHemRwas finally carried out in 20mM sodium phosphatebuffer, pH 7, 0.05 M NaCl, and 0.08% ZW 3-14.TonB116 was purified as described by Koedding et al.

with the following modification inspired by HasBpurification protocol.34 Indeed, the second cation-exchange chromatography step was replaced by a size-exclusion chromatography with a Sephacryl S-100 HP 16/60 column (GE Healthcare Life Science) equilibrated with20 mM Tris–HCl, 100 mM NaCl, and 1 mM ethylenedia-minetetraacetic acid, pH 8.Purified ferrichrome transporter FhuA was kindly

provided by Cécile Breyton. FhuA purification was asdescribed by Moeck et al.,49 except that lauryldimethyla-mine oxide (n-dodecyl-N,N-dimethylamine-N-oxide; Ana-trace) was used as a detergent instead of Triton X-100 (1%for the solubilization of the outer membrane and 0.05% forthe Ni-NTA column).Protein expression and purification were monitored with

SDS-PAGE on 15% (w/v) polyacrylamide gels stained withCoomassie Blue. All purification steps were performed at+4 °C with protease inhibitor cocktails (Roche). Themembrane transporters were purified at room temperature.HasA and HasR concentrations were measured using

their previously determined extinction coefficients (ε).50,29For the other proteins, concentrations were estimatedfrom their absorbance at 280 nm using a calculated εvalue: 10,000 M−1 cm−1 for HasB133; 8672 M−1 cm−1 forTonB116; 115,808 M−1 cm−1 for HemR; and 103,690 M−1

cm−1 for FhuA. Absorption spectra (UV–visible) wererecorded on a Perkin-Elmer lambda 2 spectrophotometerusing 1- or 0.2-cm path-length cuvettes.

Growth test

C600ΔhemA and C600ΔhemAtonB trp::Tn10 harboringthe HasRΔtbb mutant plasmid were used for growth testswith exogenous heme and heme-loaded hemophore asdescribed by Létoffé et al.51

Analytical ultracentrifugation

Ultracentrifugation studies were performed in an XLA(Beckmann) analytical ultracentrifuge using 12-mm path-length cells. Purified HasB samples were 3×10−5 and1×10−4 M in 50 mM sodium phosphate, 50 mM NaCl, pH7, buffer. The sedimentation/diffusion equilibrium wasachieved after 19 h at 18,000 rpm. The absorbance in thecell was scanned as a function of the distance to therotation axis at 277 nm for the 3×10−5 M sample and at304 nm for the 1×10−4 M sample. Experiments wereperformed at 20 °C. Data were analyzed by means ofDATA-RED software provided with the XLA centrifuge.

Isothermal titration calorimetry

Titrations were performed at 25 °C using a MicroCal VPtitration calorimeter (MicroCal Inc., Northampton, MA).All of the samples were extensively dialyzed in the bufferand thoroughly degassed before use by stirring undervacuum. All injections were carried out at 4-min intervals.

849Signal Transduction by an Outer Membrane Transporter

Titrations were carried out by injecting 20–35 consecu-tive aliquots (5–10 μL) of HasB133 or TonB116 of variousconcentrations into the ITC cell containing the transporter.A table of sample concentrations used for ITC titration ispresented in the Supplementary Material (TS1). Thebuffers were 20 mM sodium phosphate, pH 7, 0.08%ZW 3-14 in the presence of HasR; 20 mM sodiumphosphate buffer, pH 7, 0.05 M NaCl, 0.08% ZW 3-14 forthe experiments carried out with HemR; and 20 mMsodium phosphate, pH 7, 0.05% lauryldimethylamineoxide (Anatrace) for titration with FhuA.The heat of dilution of HasB133 and TonB116 injections

was determined either by injecting the ligand into thebuffer alone or by injecting more ligand into the cell aftersaturation. The value obtained was subtracted from theheat of reaction to give the effective heat of binding. Theresulting titration data were analyzed using the ORIGINsoftware package provided by the manufacturer. Themolar binding stoichiometry (N), association constant (Ka;Kd=1/Ka) and binding enthalpy change (ΔH) weredetermined by fitting the binding isotherm to a modelwith one set of sites. For the fit, any constraints instoichiometry and ΔH were not fixed. Changes in freeenergy (ΔG) and entropy changes (TΔS) were calculatedfrom ΔG=−RT ln Ka=ΔH−TΔS, where R is the gasconstant and T is the temperature in kelvins. All values arethe average of those obtained by three experiments ± thestandard deviation (SD) of the mean. Errors in TΔS valueswere calculated as [SD2(ΔH)+SD2 (ΔG)]1/2.ΔΔH was calculated as ΔH[(substrate+apoHasR)+

HasB133]−ΔH(apoHasR+HasB133), andΔ(TΔS)was calcu-lated in the same manner.ITC titration of HasB133 and TonB116 with either HasA

or membrane preparation containing HasR did not showany enthalpic signal of interaction.

Acknowledgements

We thank Cécile Wandersman for fruitful discus-sions and interest in our work. We gratefullyacknowledge Wolfram Welte and Najla Benevides-Matos for providing TonB116 and HemRhis plas-mids, respectively, and Cécile Breyton for purifiedFhuA. We thank Thierry Rose for the analyticalultracentrifugation experiment and Sylviane Hoosof the Plate-forme de Biophysique (Institut Pasteur,France) for technical assistance. Julien Lefèvre wassupported by a fellowship from the Ministère del'Education Nationale, de la Recherche et de laTechnologie (MENRT).

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.03.044

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