RESEARCH ARTICLE Open Access

Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteinsBjorn Vergauwen*, Ruben Van der Meeren, Ann Dansercoer and Savvas N Savvides

Abstract

Background: The Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires theredox-active tripeptide by import. The dedicated glutathione transporter belongs to the ATP-binding cassette(ABC)-transporter superfamily and displays more than 60% overall sequence identity with the well-studieddipeptide (Dpp) permease of Escherichia coli. The solute binding protein (SBP) that mediates glutathione transportin H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family5 SBP’s. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBPwas previously annotated as a heme-binding protein (HbpA), and was thought to mediate heme acquisition.Nonetheless, although many SBP’s have been implicated in more than one function, a prominent physiological rolefor GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought tocharacterize five representative GbpA homologs in an effort to delineate the novel GbpA-family of glutathione-specific family 5 SBPs and to further clarify their functional role in terms of ligand preferences.

Results: Lipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificitywas evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin wasobserved for all five GbpA homologous test proteins. Three out of five test proteins were found to bindglutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of thetested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequencealignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separatephylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determinedthe crystal structure of one of the GbpA family outliers from H. parasuis. Comparisons thereof with the previouslydetermined structure of GbpA in complex with oxidized glutathione reveals the structural basis for the lack ofallocrite binding capacity, thereby explaining the outlier behavior.

Conclusions: Taken together, our studies provide for the first time a collective functional look on a novel,Pasteurellaceae-specific, SBP subfamily of glutathione binding proteins, which we now term GbpA proteins. Our studiesstrongly implicate GbpA family SBPs in the priming step of ABC-transporter-mediated translocation of useful forms ofglutathione across the inner membrane, and rule out a general role for GbpA proteins in heme acquisition.

Keywords: glutathione, GbpA, HbpA, DppA, solute-binding protein, SBP, ABC transporter

BackgroundATP-binding cassette (ABC)-transporters exist in allthree kingdoms of life and transport a large variety ofsubstrates across biological membranes. In addition totheir well-documented role in solute transport, a diver-sity of sensory functions have been assigned that

implicate ABC-transporters in the maintenance of cellintegrity, responses to environmental stresses, cell-to-cell communication and cell differentiation and inpathogenicity. Based on the direction of transport, ABCtransporters can be classified as either exporters orimporters. Both classes are characterized by the couplingof two nucleotide-binding domains (NBD) and twotransmembrane domains (TMD). In the case of ABCimporters, which are found exclusively in prokaryotes, afifth domain, termed the solute binding protein (SBP), is

* Correspondence: Bjorn.Vergauwen@ugent.beUnit for Structural Biology, Laboratory for Protein Biochemistry andBiomolecular Engineering (L-ProBE), Department of Biochemistry andMicrobiology, Ghent University, 9000 Ghent, Belgium

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

© 2011 Vergauwen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

part of the functional unit [1]. SBPs bind their ligandswith high affinity and deliver them to the permease unit(the TMDs), where the substrate is released into thetranslocation pore upon ATP binding and hydrolysis inthe NBDs [2,3]. SBPs are located in the periplasm ofGram-negative bacteria, or lipid-anchored to the cellwall, or fused to the TMD in the case of Gram-positivebacteria and Archaea [4]. Although SBPs of Gram-nega-tive bacteria exist predominantly as stand-alone peri-plasmic proteins, they are sometimes connected in afusion protein with the TMD [4] or observed lipid-anchored to the inner membrane [5,6]. The physiologi-cal relevance of the immobilized versions of SBPsremains largely unaddressed in the literature.Based on sequence homology analyses, the bacterial

SBP superfamily has been classified into 8 clusters, withcluster 5 comprising dipeptide binders (DppA family),oligopeptide binders (OppA family) and nickel specificSBP’s (NikA family) [7]. Continuous family updates bythe Transporter Classification Database http://www.tcdb.org has now led to a cluster 5 SBPs containing up to 27different subfamilies that are associated with transloca-tion cargos as diverse as - in addition to di-and oligo-a-peptides and nickel substrates - antimicrobial peptides,δ-aminolevulinic acid, heme, plant opines, carbohy-drates, the osmoprotective proline betaine, and themetal-chelater ethylene diamine tetraacetate. The mostrecent addition to the SBP family is termed GbpA(TCID: 3.A.1.5.27), a lipoprotein from Haemophilusinfluenzae, which binds reduced (GSH) and oxidized(GSSG) forms of glutathione to prime the dipeptide-DppBCDF ABC-transporter for glutathione translocationacross the inner membrane [8]. Structural studies of thehighly homologous GbpA from H. parasuis in complexwith GSSG have revealed structural features that typifycluster 5 SBPs, namely, a pear-shaped, two-domain a/b-fold that collapses about the hinge region connectingthe N-and C-terminal domains to sandwich the molecu-lar cargo, in this case a single GSSG molecule [8]. In theabsence of ligand, SBP’s are flexible with the twodomains rotating around the hinge and existing largelyin the open conformation with both domains separated.Substrate binding induces the closed conformation, andthe ligand is trapped at the interface between the twodomains, according to what has been termed the “VenusFly-trap” mechanism [9]. The structural analysis ofGbpA in complex with GSSG has identified many speci-fic interactions between GSSG and its cognate SBP thatmay be helpful in the delineation of the entire GbpAfamily [8]. The discovery that GbpA mediates glu-tathione transport in H. influenza came as a completesurprise as this protein was previously thought to be aheme-binding protein, accordingly annotated HbpA, andwas implicated as a binding-platform for heme [5,10].

Nonetheless, GbpA does bind hemin, albeit weakly withan apparent Kd of 655 μM [8], and a possible role forGbpA and DppBCDF in heme acquisition has beendescribed [10,11]. In this regard, GbpA presents itself asa good example of the high degree of substrate promis-cuity especially common among cluster 5 SBPs [12-15].In light of our recent report on the functional reanno-

tation of HbpA to GbpA [8], the present study wasdesigned to elucidate further and refine this emergingSBP subfamily of glutathione-binding proteins and toclarify the roles of such proteins in glutathione andheme acquisition. GbpA homologs were identifiedemploying BLAST and their clustering in the novelGbpA family was established based on structure-basedmotif fingerprinting. To ascertain the GbpA family func-tionally, we subsequently explored the ligand prefer-ences of five representative GbpA homologous proteins.As the GbpA from H. influenzae is lipidated in vivo, wealso incorporated in our test protein set GbpA homolo-gous sequences that were not preceded by a peptidase IImodifiable leader peptide, thereby providing the oppor-tunity to uncover lipidation-dependent functionaleffects. Our studies indicate that GbpA family membersare exclusively found in the Gram-negative Pasteurella-ceae, where they have evolved by gene duplication froma canonical DppA sequence to prime the transport ofphysiologically useful forms of glutathione. Our data onthe other hand do not support a general role for GbpAfamily proteins in heme acquisition. Finally, a phylogen-tically distinct cluster of GbpA homologues was identi-fied, which appears to lack binding capacity not only forglutathione and other peptide ligands, but heme as well,thus casting a new twist in the possible substrate prefer-ences of GbpA-like proteins.

Results and DiscussionIn order to delineate the GbpA family of SBP proteinsand to identify GbpA homologs with signal peptidaseII modifiable leader peptides, we BLASTed the GbpAHi

sequence against all available microbial databases inJune 2011 http://www.ncbi.nlm.nih.gov/sutils/genom_-table.cgi. We found GbpA homologs in 13 differentspecies, all of which belong to the Pasteurellaceae, andmore than half of these sequences (belonging to 7 spe-cies) were predicted lipoproteins by the LipoP 1.0 ser-ver http://www.cbs.dtu.dk/services/LipoP/. A survey ofthe top 100 homologs furthermore uncovered a num-ber of established and predicted DppA proteins as wellas several Pasteurellaceae-unique sequences that onfirst sight neither belong to the DppA-family, nor tothe GbpA-family and that are all annotated as heme-binding proteins (HbpA). We will refer to these pro-teins with the affiliation HbpA2. These HbpA2sequences were found in 3 species, H. parasuis, M.

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 2 of 10

haemolyticus, and A. pleuropneumoniae, all of whichalso contained a GbpA. The identified GbpA, DppA,and HbpA2 proteins share at least 50% sequence iden-tity, with the GbpA/HbpA2 couples being the closestrelatives (all exceeding 60% sequence identity) and theDppA/HbpA2 couples having the most distant rela-tionship. A phylogentic analysis using the Geneious5.3.4 package http://www.geneious.com led to a strik-ing delineation of these homologs into three clades, assupported by bootstrap resampling (Figure 1). Interest-ingly, most DppA proteins homologous to GbpAHi arealso found within the Pasteurellaceae, strongly suggest-ing that glutathione-specific GbpA proteins evolvedparalogously in the Pasteurellaceae lineage from theircanonical DppA dipeptide-binder. High-resolutioncrystal structures of liganded GbpA (in complex withGSSG) and DppA (in complex with glycylleucine)representatives have uncovered key ligand contact resi-dues that provide family-specific signature sequences[8,16]. We highlighted such sequence fingerprints in acut-and-spliced version of a hierarchical clustering-based multiple sequence alignment http://multalin.

toulouse.inra.fr/multalin/ of the GbpAs’ BLAST top100 homologs shown in Figure 2. This analysis corre-lates strongly with the three clade separation, andreveals a strict conservation of 13 out of 18, and 8 outof 10 of the active site residues in the demarcatedGbpA and DppA clade, respectively. Furthermore, Fig-ure 2 highlights the versatility of the dpp-fold wherebya handful of key mutations on either side of the bind-ing interface has led to a ligand-preference switchfrom a dipeptide to a disulfide bridge containing hexa-peptide. Accordingly, the signature sequence for theGbpA family is more extended and comprises moreresidues than that of the DppA family.Interestingly, the HbpA2 clade diverged significantly

from both the GbpA and DppA signature sequences. Infact, some of the strictly conserved residues that contactthe ligand’s charged N- and C-termini in either theGbpA or the DppA family are replaced by physico-chemically dissimilar residues in the HbpA2 sequencesthereby virtually disrupting critical ligand-stabilizing saltbridges (In case of GbpA-GSSG binding, Arg33 substi-tuted by a Thr, and Asp432 substituted by an Arg; in

Figure 1 Phylogenetic analyses of the top 100 GbpA homologs found in the National Center for Biotechnology Information (NCBI)microbial protein database reveal three distinct branches, clustering GbpA SBP’s, canonical DppA proteins, and HbpA2 SBP’s. Thephylogenetic tree was generated using the neighbor-joining tree construction method with Jukes-Cantor distances within the Geneious 5.3.4.software program and no outgroup was selected. Bootstrap resampling was conducted with 100 replicates by PhyML 3.0 [30] and supportvalues for the three main nodes are provided. Representative sequences are shown by their NCBI Reference Sequence identifier.

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 3 of 10

case of DppA-dipeptide binding, Asp408 substituted byan Arg). The ligand specificity of the HbpA2 clade istherefore difficult to predict, but it is highly unlikelythat glutathione or dipeptides are the natural molecularcargos. Given the auxotrophic nature of Pasteurellaceaefor heme and the fact that the dpp-architecture is a pro-ven hemin-binding scaffold (E. coli DppA binds heminwith a 10 μM affinity [14] and also GbpAHi displays an,albeit low, affinity for hemin [8]) it is tempting to specu-late that HbpA2 proteins may play a role in hemetransport.

To document the heme-binding characteristics of theGbpA family, to verify the role of the posttranslational1,2-diacylglycerol-modification of GbpA proteins interms of glutathione and heme binding, and to establishthe ligand-preferences of the HbpA2 family, we selectedin addition to GbpAHi yet another GbpA lipoprotein(from A. pleuropneumoniae, GbpAAp), 2 non-lipoproteinGbpA’s (GbpAHp and GbpAPm from H. parasuis and P.multocida, respectively), and 2 HbpA2 proteins(HbpA2Hp and HBPA2Ap from H. parasuis and A. pleur-opneumoniae, respectively), for further study.

Figure 2 Cut-and-spliced version of a hierarchical clustering-based multiple sequence alignment of the top-100 GbpA homologsfound in the NCBI microbial protein database reveal invariant signature sequences for the GbpA and DppA subfamily. Green-boxedresidues of GbpA family members are ligand-interacting residues identified from the GSSG/GbpAHp complex crystal structure (pdb id. 3M8U).Purple-boxed residues of DppA family members interact with the dipeptide allocrite in the E. coli DppA/glycylleucine complex structure (pdb id.1DPP). Sequence names are NCBI Reference Sequence identifiers. The numbering on the top corresponds to the GbpAHp sequence, while thenumbering at the bottom refers to the DppA sequence of E. coli.

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 4 of 10

In a previous report, we had employed a hemin-bind-ing assay based on native-PAGE to show that GbpAHi

has a physiologically irrelevant affinity for hemin [8]. Toprobe heme-binding among our protein test panel, thepurified recombinant soluble forms of the test proteinswere subjected to our native-PAGE-based assay in thepresence and absence of 0.5 mM hemin (Figure 3). Asthis hemin concentration approaches its Kd-value, theGbpAHi band splits up, with about half of it migratingfaster because of complexation with hemin (as judgedby visual inspection (red-brownish bands) and heme-staining with 2,3’,5,5’-tetramethylbenzidine/H2O2).Although all tested proteins displayed the split migra-tion pattern, the fraction of the faster running hemin-complexed bands is much lower compared to that ofGbpAHi and therefore indicative of an extremely lowaffinity for hemin. These results strongly suggest thatheme-binding is not a general feature of the GbpA andHbpA2 family. Notably, the second best binder ofhemin is GbpAAp, the other lipoprotein in our testpanel.A fluorescence-based thermal shift assay (Thermo-

fluor assay [17]) was subsequently employed to screenfor potential allocrites. The set of putative ligandsamounted to 15 different ligands comprising of glu-tathione, some of its derivatives, and already estab-lished allocrites of the type 5 SBP superfamily such asdi- and tripeptides, δ-aminolevulinic acid, nickel, andproline-betaine. The temperature-induced changes inrelative fluorescence of 100 μg of test protein as afunction of candidate ligands at 1 mM were recordedand those ligands that significantly affected the transi-tion midpoint temperature (Tm) of the apo-form(threshold was set at 1.5°C) are shown in Table 1 as afunction of the corresponding ΔTm-values (apparentTm-differences in °C for the ligand-protein complexesrelative to the uncomplexed proteins). Our analysisrevealed an affinity of all tested GbpA proteins for(ranked according to descending ΔTm): GSSG > GSH> S-methylglutathione ≅ glutathione-cysteine disulfide.

In addition, GbpAHp and GbpAPm also showed aminor but significant Tm-shift in the presence of thebulky S-alkylated glutathione derivatives, S-hexylglu-tathione and S-decylglutathione. Although certain S-modifications were tolerated, fragments of glutathionesuch as g-glutamylcysteine or cysteinylglycine or aslightly elongated form of glutathione (homoglu-tathione) did not influence the melting behavior of anyof the tested proteins, showing that the GbpA familycarries a specificity for the glutathione backbone. Inter-estingly, in contrast to the notion that increasinglybulkier S-alkylations abrogate binding, the disulfide ofglutathione with another glutathione molecule or withcysteine appear to be good allocrites for the entireGbpA family, strongly suggesting that the GbpA-foldevolved to bind these types of glutathione derivativesin vivo. This observation makes sense as many Pasteur-ellaceae are glutathione as well as cysteine auxotrophsand glutathione-cysteine disulfide reaches levels similarto those of glutathione in human plasma (up to 10 μM[18]).Isothermal titration calorimetry (ITC) was subse-

quently used to determine the equilibrium dissociationconstants for the interaction of our GbpA proteins withGSSG, GSH, and S-me-GSH. Typical ITC thermograms,showing the raw and integrated data for the interactionof GbpAHp with these allocrites are shown in Figure 4,and all respective calculated Kd-values are summarizedin Table 2. Except for GbpAHp, the ranking of bindingstrength according to the thermal shift ΔTm-values wasrecapitulated by the ITC-derived Kd-values. Notably,affinities for the natural allocrites, GSSG and GSH, var-ied 200-fold, and for the artificial ligand S-me-GSH ~400-fold among the selected GbpA-family members,with GbpAHi being the worst binder for all tested puta-tive ligands. Interestingly, GbpA from H. influenzae,which naturally exists in a membrane anchored form,takes a unique position within the GbpA-family as thebest binder of hemin, and the worst binder of glu-tathione. On the other hand, the soluble form of thepredicted lipoprotein GbpA from A. pleuropneumoniaedisplays affinities for the tested glutathione derivativesthat are similar to the two non-lipoprotein GbpA’s (seeTable 2). Therefore, membrane-anchoring of GbpA pro-teins appears not to impose any functional implications.Interestingly, the best hemin-binders from our test pro-teins were the lipoprotein GbpAs (Figure 3), amongstwhich the one of H. influenzae was shown to be biologi-cally significant for heme acquisition [10,11]. Therefore,membrane-anchoring may influence the role of GbpAsin heme acquisition by increasing their intrinsic affinityfor hemin. Nonetheless, GbpA-mediated heme importappears to be of minor importance under laboratoryconditions as yet another family 5 SBP, the antimicrobial

Figure 3 Characterization of the hemin-binding properties ofGbpA and HbpA2 family members. Native-PAGE analysis of 10 μgtest protein in the absence (-) or the presence (+) of 0.5 mM hemin.The faster migrating band for each test protein in the presence ofhemin represented the hemin/protein complex as verified by heme-staining (see Materials).

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 5 of 10

peptide binder SapA, has recently been shown to beessential for heme utilization by iron-starved nontype-able H. influenzae cells [19].The lack of conservation of consensus sequence fin-

gerprints important for ligand-binding by GbpA- and

DppA-family proteins (Figure 2) had already suggestedthat the HbpA2 proteins in our test set would fail tobind the glutathione- and dipeptide-types of ligands.Indeed, our thermofluor analyses showed that none ofthe two HbpA2 proteins under study were able to

Table 1 Summary of results obtained from thermal shift assays for the identification of GbpA- and HbpA2-familyligands out of a test set of typically family 5 SBP allocrites.

GbpAHi GbpAAp GbpAHp GbpAPm HbpA2Hp HbpA2Ap

Tm (°C)a

GSSG 17.5 14.0 16.0 20.5 - -

GSH 12.5 11.0 14.0 13.0 - -

S-methylglutathione 10.5 7.5 11.5 11.5 - -

S-nitrosoglutathione 7.0 6.5 9.5 12.0 - -

glutathione cysteine disulfide 10.0 6.0 7.0 12.0 - -

S-hexylglutathione - - 4.5 6.5 - -

S-decylglutathione - - 2.5 3.0 - -

homoglutathioneg-glutamylcysteinecysteinylglycineglycylleucineglycylglycylcysteineδ-aminolevulinic acidproline-betainenickel}

- - - - - -

aInteracting ligands are identified by means of the respective ΔTm-values, the apparent Tm-differences in °C for the ligand-protein complexes relative to theuncomplexed proteins. A negative sign indicates ligands for which the ΔTm is smaller than 1.5°C.

Figure 4 Determination of the affinity constant of GbpAHp for three different glutathione forms: the oxidized (GSSG), the reduced(GSH), and an S-derivatized form (S-me-GSH). Isothermal titration data for the titration of GbpAHp with either GSSG, GSH, or S-me-GSH in 10mMTris-HCl, pH 7.4. The upper panels show the calorimetric titrations for 10-μl injections with 300 s between injections. The lower panelsrepresent the integrated heat values (from the upper panels) as a function of the protein/ligand molar ratio in the cell. The solid line representsthe best fit of the single-site model to the experimental points.

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 6 of 10

interact with any of the tested type 5 SBP superfamilyallocrites (Table 1). Because of the possibility that theHbpA2 proteins would co-purify with their naturalligands, as observed for some other structurally charac-terized SBP’s, such as e. g. the cysteine-complexed CjaAfrom Campylobacter jejuni [20] or the oligopeptide-bin-der AppA from Bacillus subtilis in complex with a non-apeptide [21], we sought to determine the crystalstructure of HbpA2Hp hoping to elucidate an interactionwith a possible ligand. The crystal structure of HbpA2Hp

was determined to 2.0 Å resolution by maximum-likeli-hood molecular replacement (Figure 5; additional file 1,Table S1). The structure reveals the two-lobe a/b-foldarchitecture and b-strand topology typical for SBP-likeproteins, and is essentially identical to that of the struc-turally characterized GbpA and DppA proteins. Crystal-lographic refinement and exhaustive examination ofresidual difference electron density maps failed to pro-vide any evidence for a bound ligand to HbpA2. More-over, the N- and C-terminal domains were opened byabout 33 degrees with respect to the GSSG-boundGbpA and glycylleucine-bound DppA reported pre-viously [8,16] (Figure 5A), again indicating we crystal-lized apo-HbpA2Hp. Importantly, the crystal structure ofHbpA2Hp offers an explanation for its inability to bindpeptide-like ligands. Figure 5B shows a structural super-position of residues of the GbpA ligand-binding sitewith only those corresponding residues in HbpA2Hp ofwhich the physicochemical properties are significantlydifferent as revealed by our sequence alignments (Figure2). This analysis focuses on the C-terminal lobe, becauseit comprised the majority of the ligand-interacting resi-dues as shown by the GbpAHp-GSSG complex [8] (13out of 18 interactions), and due to the fact that it isbelieved to drive formation of the SBP-ligand-encountercomplex [22]. Out of the 13 GSSG-contacting residues,3 were not strictly conserved in HbpA2Hp, i.e. A380P,S430T, and D432R. All of these residues appear to becritical for GSSG-binding by GbpAHp: the peptide-nitro-gen of A380 hydrogen-bonds with the carbonyl oxygenof GlyI of one of the glutathione legs (GS-I); the D432

side chain carboxylate forms a salt bridge with theamino terminus of GS-I as well as H-bonds with theside chain hydroxyl groups of Y138 and Y521 therebypositioning these residues for favorable hydrophobicinteractions, the side chain of S430 is involved in H-bonding with both the carboxylate- and amino-groupsof the g-glutamyl-moiety of GS-I [8]. The structuralsuperposition in Figure 5B shows that S430 and D432 inthe HbpA2Hp structure occupy the exact same positionas the corresponding active site residues in GbpAHp. At

Figure 5 Structure of HbpA2 from H. parasuis. (A) Ribbondiagram showing an overlay of GSSG-complexed GbpA (PDB id.3M8U; gray) with HbpA2 from H. parasuis (PDB id. 3TPA; blue). Thestructures were superposed with respect to their C-terminaldomains. HbpA2 shows a conformation that opens the cleftbetween the N- and C-terminal domains about 30° relative to itsligand-complexed paralogous counterpart. GSSG is depicted inatom-colored sticks. (B) Key binding residues of the GbpA C-terminal domain to accommodate GSSG (shown in atom-coloredgray sticks) are replaced in HbpA2 by counterparts (shown in atom-colored blue sticks) that are incompatible with binding peptide-likeallocrites. Residue numbering is according to PDB id. 3M8U. Somekey interactions are depicted as black dashed lines. For clarity someinteractions have been omitted. The figure was created with PyMOL(The PyMOL Molecular Graphics System, Schrödinger, LLC).

Table 2 Summary of the dissociation constants for theinteraction of our GbpA-family test set with thephysiologically relevant glutathione forms (GSSG andGSH), and the artificial S-methylglutathione (S-me-GSH)as determined by ITC at 37°C.

GbpAHia GbpAAp GbpAHp GbpAPm

Kd (μM)

GSSG 12.9 ± 0.3 0.33 ± 0.05 2.1 ± 0.2 0.15 ± 0.03

GSH 56.4 ± 3.0 1.58 ± 0.2 1.9 ± 0.1 0.26 ± 0.05

S-methylglutathione 212 ± 17 1.17 ± 0.05 0.90 ± 0.07 0.55 ± 0.04aValues were taken from ref. [8].

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 7 of 10

the same time, A380 takes a slightly different positionwhich would be expected due to the elimination of thespecial structural role of a proline residue in maintain-ing loop structure at this position. Our structural analy-sis offers direct evidence that the A380P, S430T, andD432R substitutions would be grossly incompatible withGSH and GSSG binding as they would abolish electro-static, H-bonding and hydrophobic interactions contri-butions critical for binding of such ligands. A similaranalysis, this time against E. coli DppA, shows that R415in HbpA2Hp takes the exact same position as the activesite residue D408 in E. coli DppA, a residue that makesa salt-bridge with the amino-terminus of the bounddipeptide ligand. Thus, R415 would prevent dipeptideligand binding. Finally, we note that the inability of theHbpA2 proteins to interact with either glutathione ordipeptides is correlated by looking at the interspeciesoccurrence: 2 out of 3 species with HbpA2 genes alsocarry genes for both a GbpA and a DppA familymember.

ConclusionsWe here have provided a biochemical and phylogeneticdelineation of the GbpA-family of glutathione-bindingproteins. We showed that the GbpA proteins likelyevolved exclusively within the Pasteurellaceae lineage bygene duplication from an already present dipeptide-binding protein, DppA, thereby explaining our pre-viously reported functional annotation of GbpA proteinsas periplasmic binding proteins that prime glutathioneimport to the cytoplasm via the cognate Dpp-ABCtransporter [8]. GbpA proteins are specific for the glu-tathione backbone, but can tolerate S-modifications todifferent extends. This slightly promiscuous behaviorprobably resulted from the evolutionary tailoring of theGbpA scaffold to also accommodate useful disulfides ofglutathione, such as GSSG and glutathione cysteine dis-ulfide. Although GbpA proteins were formerly known asheme-binding proteins, an important implication of ourwork concerns the awareness that they clearly do nothave a general role in heme acquisition. Apart fromGbpA and/or DppA representatives, some Pasteurella-ceae also carry the genetic information for a close,although phylogenetically distinct homolog, which wehave termed HbpA2 in the present paper. Because wewere unable to identify a molecular interaction partnerfor these paralogous HbpA2 proteins, their in vivo rolewill have to await further study. In any case, the currentannotation as “heme-binding protein or HbpA” forHbpA2-family members is clearly inaccurate and data-bases should be rectified accordingly (e.g family 5 SBPwith no known function).

MethodsStrainsWild-type strain H. influenzae Rd was purchased from theAmerican Type Culture Collection (Manassas, Va.). The P.multocida and A. pleuropneumoniae clinical isolates usedin this study were a kind gift of Dr. Mario Vaneechoutte(Deptartment of Clinical Chemistry, Microbiology, andImmunology, University Hospital, Ghent, Belgium). TheH. parasuis strain used in this study was isolated from thenasal cavity of a clinically healthy pig and was kindly pro-vided by Dr. Filip Boyen (Department of Pathology, Bac-teriology and Avian Diseases, Faculty of VeterinaryMedicine, Ghent University, Belgium).

Production and purification of recombinantSBP’sThe construction of the expression plasmids pET-GbpAHi and pET-GbpAHp is described in ref. [8]. Theremaining proteins in this study were overexpressedusing similarly constructed plasmids also based on thepET20b vector template. The leader peptides of therespective proteins were predicted using the SignalP 3.0server http://www.cbs.dtu.dk/services/SignalP/ and theLipoP 1.0 server http://www.cbs.dtu.dk/services/LipoP/and replaced by the PelB leader peptide provided by thepET20b plasmid. In case of the A. pleuropneumoniaegbpA gene sequence, the codon that translates to the N-terminal Cys was furthermore replaced by the Metcodon. Therefore the mature proteins started at posi-tions 23, and 22 for the P. multocida, and A. pleurop-neumoniae GbpA family members, respectively, and atpositions 19, and 21 for the HbpA2 SBP’s of H. para-suis, and A. pleuropneumoniae, respectively. All test pro-tein coding sequences were extended with a his-tag tofacilitate purification. The respective genes were PCR-amplified using forward and reverse primers (5’ to 3’),respectively - with the cloning (restriction) site under-lined and identified between brackets: GbpAPm

(CCATGGATAATAAAACCTTTATTAACTGC [NcoI],GCGGCCGCATCCGCTAACTTAGTGC [NotI]);GbpAAp (CCATGGATGATAAAAATGCGGACG[NcoI], GCGGCCGCGTCGGCTAATTTTGTACCG[NotI]); HbpA2Hp (GATATCTCGGCACCGACAAATA-CATTG [EcoRV], CTCGAGTTAAGGCTTCAGACT-TACGCCAT [XhoI]); HbpA2Ap (CCATGGCAGCGCCGGCACATACTTTAG [NcoI], GCGGCCGCTTCCGTTAGACTCACATTATAG [NotI]).The proteins were expressed in E. coli and purified

using a three-step chromatographic protocol (IMAC,followed by anion-exchange, and size-exclusion chroma-tography) as described in ref. [8]. The concentration ofpurified proteins was determined by the Bio-Rad ProteinAssay with bovine serum albumin as the standard.

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 8 of 10

Native PAGE heme-binding gel shift assayHemin stock solutions were prepared by dissolvingbovine hemin chloride (Sigma-Aldrich) in 100 mMNaOH prior to 10-fold dilution in double distilledwater. These solutions were then neutralized to pH 7.5using HCl and filtered through a Millex-GP 0.22 μm fil-ter unit (Millipore). Stock concentrations were deter-mined spectrophotometrically (ε385 = 58,400 M-1 cm-1),and the solutions were used within a day after prepara-tion. Purified test protein (10 μg) was incubated with 0.5mM hemin or water alone for 45 min at room tempera-ture and subjected to native PAGE as described pre-viously [8]. Hemin-complexed bands were visualized in-gel by their intrinsic peroxidase activity using 2,3’,5,5’-tetramethylbenzidine and H2O2 [23]. The hemin-com-plexed protein species migrated faster compared to theapo-forms as was already described for other hemin-binding proteins [8,19,24].

Thermal denaturation assaysThermofluor thermal shift assays were conducted in aC1000 thermal cycler equipped with a CFX96 optical reac-tion module (Bio-Rad). The microplate wells were loadedwith 25-μL solutions, containing 100 μg test protein, 2 ×Sypro orange (Molecular Probes), and 1 mM of the testchemicals in 10 mM Tris-HCl, pH 8.0. The plates weresealed with Microseal B film (Bio-Rad) and heated from30°C to 90°C at a rate of 2°C per min. The unfolding reac-tions were followed by simultaneously monitoring therelative fluorescence (FRET settings) using the charge-coupled device camera. The inflection point of the fluores-cence versus-temperature curves was identified by plottingthe first derivative over the temperature, and the minimawere referred to as the melting temperatures (Tm).

Isothermal titration calorimetry (ITC)Experiments were carried out using a VP-ITC MicroCa-lorimeter (MicroCal) at 37°C, and data were analyzedusing the Origen ITC analysis software package suppliedby MicroCal. Purified test proteins were dialyzed over-night against 10 mM Tris-HCl, pH 7.4, at 4°C. Theresultant dialysis buffer was then used to dissolve thetest compounds. Protein concentrations were measuredspectrophotometrically using the respective theoreticalextinction coefficients at 280 nm as calculated from themature protein sequences at http://web.expasy.org/prot-param/. GSSG concentrations were determined by theabsorbance change at 340 nm resulting from the glu-tathione reductase-catalyzed NADPH-dependent conver-sion of GSSG to 2GSH (ε340 = 6,200 M-1 cm-1). GSHconcentrations were determined by the reaction withEllman’s reagent (ε412 = 14,000 M-1 cm-1). All solutionswere degassed prior to use. Titrations were always

preceded by an initial injection of 3 μL and were carriedout using 10-μL injections applied 300 s apart. The sam-ple was stirred at a speed of 400 rpm throughout. Testcompounds were always injected into the HbpA-con-taining sample cell. The heats of dilution were negligiblysmall for the titration of each ligand into buffer; hencethe raw data needed no correction. The thermal titra-tion data were fit to the one binding site model todetermine the dissociation constant, Kd. Several titra-tions were performed to evaluate reproducibility.

Crystallization and structure determination of HbpA2from H. ParasuisCrystallization of HbpA2Hp (10 mg/mL in 10 mMTris-HCl pH 8.0, 100 mMNaCl) was screened using a Mos-quito crystallization robot (TTP LabTech) based on 200nL crystallization droplets (100-nL protein sample and100-nL crystallization condition) equilibrated in sitting-drop geometry over 25-μL reservoirs containing a givencrystallization condition. This led to the development ofalready well-formed rod-shaped crystals in condition 39of the Hampton Research Index screen (0.1 M HEPESpH 7.0,30% v/v jeffamine ED-2001). This condition wasoptimized using a bigger “sitting-drop” geometry as fol-lows. Crystallization droplets consisting of 1-μL proteinsample and 1 μL 0.1 M HEPES pH 7.0, 30% v/v jeffamineED-2001, were equilibrated against 0.75-mL reservoirsolution containing 5-20% wt/v saturated ammoniumsulfate. Diffraction quality crystals of HbpA2Hp grewovernight as clusters of easy separable crystalline rods(measuring 0.05 × 0.05 × 0.2 mm). For data collectionunder cryogenic conditions (100 K), single crystals wereflash cooled with the help of a nylon loop directly inliquid nitrogen after a very brief incubation (typically <30 s) in cryoprotecting solution containing 0.1 M HEPESpH 7.0, 30% v/v jeffamine ED-2001, and 20% v/v glycerol.The structure of HbpA2 from H. parasuis was deter-mined by maximum-likelihood molecular replacement asimplemented in the program suite PHASER [25]. Thesearch model was prepared from the structure ofH. parasuis GbpA in complex with GSSG [8] using theprogram Chainsaw [26], based on structure-basedsequence alignments. The final search model containedalanines at all nonconserved positions and was strippedfrom all solvent molecules and ligand. The best solutionwas obtained in a combined search strategy whereby wesearched for the C-terminal domain first. Inspection ofelectron density maps calculated with Fourier coefficients2Fo-Fc, MR, ac, MR confirmed the solution as evidenced byextra density for missing structural elements and sidechains. Model (re)building was carried out via a combina-tion of automated methods as implemented in the PHE-NIX suite [27] and manual adjustments using the

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 9 of 10

program COOT [28]. Crystallographic refinement andstructure validation was carried out using PHENIX andBuster [27,29].

Additional material

Additional file 1: Table S1. X-ray data collection and refinementstatistics for HbpA2 of H. parasuis.

AcknowledgementsThis work was supported by Grant 3G020506 to BV and Grant 3G064307 toSNS via the Research Foundation Flanders, Belgium (FWO). BV and RVDM arepostdoctoral and predoctoral research fellows of the FWO, respectively. Wethank the Swiss Light Source (Villigen, Switzerland) for synchrotronbeamtime allocation and the staff of beamline PXIII for technical support.We express our gratitude to Annelies Van Raemdonck for excellent technicalassistance.

Authors’ contributionsBV designed the study and was involved in all experimental aspects of thework. AD contributed to recombinant protein production. RVdM and SNScarried out crystallographic studies. BV and SNS supervised the work andwrote the manuscript. All authors have read and approved the finalmanuscript.

Received: 12 September 2011 Accepted: 16 November 2011Published: 16 November 2011

References1. Higgins CF: ABC transporters: from microorganisms to man. Annu Rev Cell

Biol 1992, 8:67-113.2. Wen PC, Tajkhorshid E: Conformational Coupling of the Nucleotide-

Binding and the Transmembrane Domains in ABC Transporters. Biophys J2011, 101:680-690.

3. Oldham ML, Chen J: Crystal structure of the maltose transporter in apretranslocation intermediate state. Science 2011, 332:1202-1205.

4. van der Heide T, Poolman B: ABC transporters: one, two or fourextracytoplasmic substrate-binding sites? EMBO Rep 2002, 3:938-943.

5. Hanson MS, Slaughter C, Hansen EJ: The hbpA gene of Haemophilusinfluenzae type b encodes a heme-binding lipoprotein conservedamong heme-dependent Haemophilus species. Infect Immun 1992,60:2257-2266.

6. Wyszynska A, Tomczyk K, Jagusztyn-Krynicka EK: Comparison of thelocalization and post-translational modification of Campylobacter coliCjaC and its homolog from Campylobacter jejuni, Cj0734c/HisJ. ActaBiochim Pol 2007, 54:143-150.

7. Tam R, Saier MH Jr: Structural, functional, and evolutionary relationshipsamong extracellular solute-binding receptors of bacteria. Microbiol Rev1993, 57:320-346.

8. Vergauwen B, Elegheert J, Dansercoer A, Devreese B, Savvides SN:Glutathione import in Haemophilus influenzae Rd is primed by theperiplasmic heme-binding protein HbpA. Proc Natl Acad Sci USA 2010,107:13270-13275.

9. Mao B, Pear MR, McCammon JA, Quiocho FA: Hinge-bending in L-arabinose-binding protein. The “Venus’s-flytrap” model. J Biol Chem 1982,257:1131-1133.

10. Morton DJ, Madore LL, Smith A, Vanwagoner TM, Seale TW, Whitby PW,Stull TL: The heme-binding lipoprotein (HbpA) of Haemophilusinfluenzae: role in heme utilization. FEMS Microbiol Lett 2005, 253:193-199.

11. Morton DJ, Seale TW, Bakaletz LO, Jurcisek JA, Smith A, VanWagoner TM,Whitby PW, Stull TL: The heme-binding protein (HbpA) of Haemophilusinfluenzae as a virulence determinant. Int J Med Microbiol 2009,299:479-488.

12. Berntsson RP, Doeven MK, Fusetti F, Duurkens RH, Sengupta D, Marrink SJ,Thunnissen AM, Poolman B, Slotboom DJ: The structural basis for peptideselection by the transport receptor OppA. EMBO J 2009, 28:1332-1340.

13. Eswarappa SM, Panguluri KK, Hensel M, Chakravortty D: The yejABEFoperon of Salmonella confers resistance to antimicrobial peptides andcontributes to its virulence. Microbiology 2008, 154:666-678.

14. Letoffe S, Delepelaire P, Wandersman C: The housekeeping dipeptidepermease is the Escherichia coli heme transporter and functions withtwo optional peptide binding proteins. Proc Natl Acad Sci USA 2006,103:12891-12896.

15. Dasgupta A, Sureka K, Mitra D, Saha B, Sanyal S, Das AK, Chakrabarti P,Jackson M, Gicquel B, Kundu M, Basu J: An oligopeptide transporter ofMycobacterium tuberculosis regulates cytokine release and apoptosis ofinfected macrophages. PLoS One 2010, 5:e12225.

16. Dunten P, Mowbray SL: Crystal structure of the dipeptide binding proteinfrom Escherichia coli involved in active transport and chemotaxis. ProteinSci 1995, 4:2327-2334.

17. Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E,Carver T, Asel E, Springer BA, Lane P, Salemme FR: High-densityminiaturized thermal shift assays as a general strategy for drugdiscovery. J Biomol Screen 2001, 6:429-440.

18. Kleinman WA, Richie JP Jr: Status of glutathione and other thiols anddisulfides in human plasma. Biochem Pharmacol 2000, 60:19-29.

19. Mason KM, Raffel FK, Ray WC, Bakaletz LO: Heme utilization bynontypeable Haemophilus influenzae is essential and dependent on Saptransporter function. J Bacteriol 2011, 193:2527-2535.

20. Muller A, Thomas GH, Horler R, Brannigan JA, Blagova E, Levdikov VM,Fogg MJ, Wilson KS, Wilkinson AJ: An ATP-binding cassette-type cysteinetransporter in Campylobacter jejuni inferred from the structure of anextracytoplasmic solute receptor protein. Mol Microbiol 2005, 57:143-155.

21. Levdikov VM, Blagova EV, Brannigan JA, Wright L, Vagin AA, Wilkinson AJ:The structure of the oligopeptide-binding protein, AppA, from Bacillussubtilis in complex with a nonapeptide. J Mol Biol 2005, 345:879-892.

22. Sooriyaarachchi S, Ubhayasekera W, Park C, Mowbray SL: Conformationalchanges and ligand recognition of Escherichia coli D-xylose bindingprotein revealed. J Mol Biol 2010, 402:657-668.

23. Thomas PE, Ryan D, Levin W: An improved staining procedure for thedetection of the peroxidase activity of cytochrome P-450 on sodiumdodecyl sulfate polyacrylamide gels. Anal Biochem 1976, 75:168-176.

24. Pedroche J, Yust MM, Lqari H, Megias C, Giron-Calle J, Alaiz M, Millan F,Vioque J: Chickpea pa2 albumin binds hemin. Plant Sci 2005,168:1109-1114.

25. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ:Phaser crystallographic software. J Appl Crystallogr 2007, 40:658-674.

26. Stein N: CHAINSAW: a program for mutating pdb files used as templatesin molecular replacement. Journal of Applied Crystallography 2008,41:641-643.

27. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ,Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al: PHENIX: a comprehensivePython-based system for macromolecular structure solution. ActaCrystallogr D Biol Crystallogr 2010, 66:213-221.

28. Emsley P, Cowtan K: Coot: model-building tools for molecular graphics.Acta Crystallogr D Biol Crystallogr 2004, 60:2126-2132.

29. Blanc E, Roversi P, Vonrhein C, Flensburg C, Lea SM, Bricogne G:Refinement of severely incomplete structures with maximum likelihoodin BUSTER-TNT. Acta Crystallogr D Biol Crystallogr 2004, 60:2210-2221.

30. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O: Newalgorithms and methods to estimate maximum-likelihood phylogenies:assessing the performance of PhyML 3.0. Syst Biol 2010, 59:307-321.

doi:10.1186/1471-2091-12-59Cite this article as: Vergauwen et al.: Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins. BMC Biochemistry2011 12:59.

Vergauwen et al. BMC Biochemistry 2011, 12:59http://www.biomedcentral.com/1471-2091/12/59

Page 10 of 10