Glutathione (GSH) is a vital intracellular cysteine-containing tripeptide across all kingdoms of life and assumes a plethora of cellular roles. Such pleiotropic behavior relies on a finely tuned spatiotemporal distribution of glutathione and its conjugates, which is not only controlled by synthesis and breakdown, but also by transport. Here, we show that import of glutathione in the obligate human pathogen Haemophilus influenzae, a glutathione auxotrophe, is mediated by the ATP-binding cassette (ABC)-like dipeptide transporter DppBCDF, which is primed for glutathione transport by a dedicated periplasmic-binding protein (PBP). We have identified the periplasmic lipoprotein HbpA, a protein hitherto implicated in heme acquisition, as the cognate PBP that specifically binds reduced (GSH) and oxidized glutathione (GSSG) forms of glutathione with physiologically relevant affinity, while it exhibits marginal binding to hemin. Dissection of the ligand preferences of HbpA showed that HbpA does not recognize bulky glutathione S conjugates or glutathione derivatives with C-terminal modifications, consistent with the need for selective import of useful forms of glutathione and the concomitant exclusion of potentially toxic glutathione adducts. Structural studies of the highly homologous HbpA from Haemophilus parasuis in complex with GSSG have revealed the structural basis of the proposed novel function for HbpA-like proteins, thus allowing a delineation of highly conserved structure-sequence fingerprints for the entire family of HbpA proteins. Taken together, our studies unmask the main physiological role of HbpA and establish a paradigm for glutathione import in bacteria. Accordingly, we propose a name change for HbpA to glutathione-binding protein A.
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Glutathione (GSH), an L-cysteine-containing tripeptide (γ-glutamyl-cysteinyl-glycine), attains mM concentrations inside cells to mediate a diversity of cellular functions, such as protection against oxidative, xenobiotic, and metal ion stresses, the control of intracellular redox homeostasis, cell signaling, and salvage of the essential amino acid cysteine. Within the cell, glutathione exists predominantly (> 98%) in the thiol-reduced form (GSH). The remaining amounts undergo thiol oxidation to form glutathione disulfide (GSSG) and mixed disulfides with target proteins (1), and thioetherifications or -esterifications to form glutathione S conjugates. Notably, such S conjugates of glutathione are intermediates of detoxification pathways for some natural or xenobiotic electrophiles (2). Glutathione biosynthesis is achieved via the sequential action of two ATP-dependent enzymes: γ-glutamylcysteine synthase and glutathione synthase. Barring a few parasitic protozoans, glutathione is universally present in eukaryotes; while in prokaryotes it is conspicuous in aerobic Proteobacteria and some Gram-positive genera of the phyla Firmicutes and Actinobacteria (3). Whereas it is essential for growth in eukaryotes (4, 5), bacterial mutants that are impaired in glutathione biosynthesis are indistinguishable from their parents under standard laboratory condition (6–8).
Nonetheless, compounding evidence indicates that irrespective of their origin, glutathione-containing cells transport glutathione (and its conjugates) against and along concentration gradients to regulate the intra- and extracellular pools. In this regard, export of glutathione is an obligatory first step in its turnover, as γ-glutamyl transferase, the only enzyme that can initiate catabolism of glutathione, has its active site on the extracellular surface of the membrane (9). Furthermore, glutathione S conjugates that form intracellularly have to be exported to complete xenobiotic detoxificiation pathways. On the other hand, the physiological importance of glutathione uptake by animal cells is currently unclear, whereas glutathione import by unicellular yeasts and bacteria has been shown to serve as a supply of organic sulfur (4, 8, 10).
Most glutathione transporters characterized thus far are ATP-binding cassette (ABC) systems, which in their most basic form consist of four core domains: two transmembrane domains forming a pore, and two nucleotide-binding domains energizing transport by ATP hydrolysis (11). Indeed, apart from two mammalian members of the organic anion transporting polypeptide (OATP or SLC21A) family (12, 13), all glutathione and glutathione S-conjugate exporters identified in bacteria, yeast, and mammalian cells belong to the multidrug resistance protein/cystic fibrosis transmembrane conductance regulator superfamily and are characterized by diverse substrate specificities. For instance, human MRP1 and MRP2 export glutathione conjugates and GSH (14, 15), whereas the Escherichia coli CydDC exports cysteine and GSH (16).
Despite significant progress on glutathione exporters, the characterization of glutathione importers is still in its infancy. The yeast outer membrane proteins, Hgt1p from Saccharomyces cerevisiae (17) and Pgt1/spOPT1 from Schizosaccharomyces pombe (18, 19), are the only eukaryotic glutathione importers characterized thus far, and they lack any ABC superfamily signature. Instead, they belong to the oligopeptide transporter (OPT) family, which is well represented in fungi and plants and to a limited extent also in bacteria (20). These importers are medium-affinity GSH transporters (Km,GSH of ∼60 μM) and are inhibited in the presence of certain glutathione conjugates and GSSG. The only report to date on a bacterial glutathione importer focuses on the yliABCD operon of E. coli K-12, which encodes a putative ABC superfamily importer. Genetic inactivation of the yliABCD operon resulted in severely retarded growth on minimal medium plates supplemented with glutathione as a sole sulfur source (10). A unique functional feature of bacterial ABC importers is that they generally depend on a dedicated substrate-binding protein that modulates specificity for their cargo (21). In Gram-negative bacteria such proteins are located in the periplasm and are accordingly termed periplasmic-binding proteins (PBPs).
We previously reported that the Gram-negative bacterium Haemophilus influenzae, a human pathogen causing an estimated 350–700,000 deaths among children yearly (World Health Organization, www.who.org), is a cysteine and glutathione auxotrophe and acquires both molecules by import mechanisms (8). Exogenous cysteine and GSH, and their corresponding oxidized forms cystine and GSSG, can sustain comparable growth rates of H. influenzae in an otherwise sulfur-free defined medium, suggesting the presence of dedicated import mechanisms for these molecules. While H. influenzae can thrive without intracellular GSH, the tripeptide is essential for the hydrogen peroxide (H2O2) scavengase PGdx (22). In addition, aerobically grown H. influenzae cells in GSH-depleted medium show a substantial induction of antioxidants [e.g., a twofold induction in the catalase (HktE) titer] and are hypersensitive toward organic peroxides and the endogenous electrophile, methylglyoxal (23, 24). Thus, glutathione-depleted H. influenzae can experience high oxidative and disulfide stresses, which may crucially impact its ability to colonize human tissues.
The availability of such insights on the role of glutathione in the physiology of H. influenzae and the general lack of knowledge regarding glutathione import systems, coupled with the global biomedical significance of H. influenzae infections, prompted us to pursue the molecular and structural basis of glutathione import in H. influenzae Rd.
Results
Transposon Mutagenesis and Isolation of Mutants Unable to Use Glutathione as a Source of Cysteine.
The cysteine and glutathione auxotrophic phenotypes of wild-type H. influenzae Rd were exploited in a biological assay to screen a transposon insertion mutant library for candidate genes that might be involved in GSSG transport. Chemically defined minimal medium (cdMIc-medium) sustains growth only when supplemented with an organic cysteine source, such as cystine or GSSG (8). Approximately 12,000 kanamycin-resistant insertion mutants were screened for loss of the ability to grow on cdMIc plates supplemented with GSSG as a source of cysteine. These screening experiments and subsequent PCR-based identifications led to the selection of a single kanamycin-resistant mutant that was consistently unable to grow on GSSG-supplemented cdMIc plates, while being indistinguishable from the wild type on cystine-supplemented plates. This mutant was transposon-interrupted in open reading frame (ORF) HI1187. Data mining suggested that ORF HI1187 encodes a permease-like protein homologous to E. coli DppB (61% sequence identity), which forms an operon structure together with the other components of a putative dipeptide (DppBCDF) ABC importer. This is the only Dpp homologous system in the H. influenzae Rd genome and is related to the dpp operons of E. coli as has been shown in a recent phylogenetic analysis (25). The HI1184-HI1187 dpp operon structure, however, does not appear to code for a DppA, the PBP that defines specificity of Dpp ABC importers. Taken together these data led to the hypothesis that GSSG might be bound in the H. influenzae Rd periplasm by a hitherto unknown PBP responsible for delivering GSSG to a Dpp permease for transport across the inner membrane.
The Periplasmic Lipoprotein HbpA Is an Essential Component of the H. influenzae Rd Glutathione ABC Importer.
BLAST search analysis against the H. influenzae Rd genome identified two ORFs with significant similarity to the E. colidppA, namely, HI0213 (20% sequence identity at the amino acid level) and HI0853 (54% sequence identity at the amino acid level). Single insertion/disruption mutants (HI0853::Cm and HI0213::Zeo) were constructed using a homology-based insertion/disruption mutagenesis strategy and were screened for loss of the ability to grow on cdMIc plates supplemented with GSSG as an organic source of cysteine. Strain HI0213::Zeo grew well on both GSSG- and cystine-cdMIc plates, whereas strain HI0853::Cm had lost the ability to grow on GSSG-cdMIc plates. To rule out possible polar effects (which are highly unlikely because HI0853 is clearly not part of an operon structure) and to prove the exclusive involvement of HI0853 in GSSG uptake, we constructed a complemented HI0853::Cm strain. This strain replicates a pACYC184 plasmid construct carrying an intact HI0853 locus, including appropriate promoter and terminator regions, to generate plasmid pACYC853. As shown in Fig. 1, plasmid pACYC853 rescued strain HI0853::Cm with respect to GSSG utilization. Moreover, strain HI0853::Cm did not accumulate intracellular glutathione when grown on cdMIc plates containing both cystine and GSSG, whereas the complemented strain (1.9 ± 0.2 nmol per mg protein) accumulated wild-type H. influenzae Rd levels (2.1 ± 0.3 nmol per mg protein). We observed good agreement of phenotypes with respect to catalase induction and methylglyoxal sensitivity for the HI0853::Cm cells grown in the presence of GSSG and wild-type cells grown in the absence of GSSG (Fig. S1). These results show that ORF HI0853 together with the dppBCDF operon encodes the components of the GSSG importer in H. influenzae Rd. Moreover, the complete loss of growth of either the HI1187 transposon mutant or the HI0853::Cm insertion mutant demonstrates that the newly identified GSSG ABC importer is the only route available to H. influenzae for GSSG take up via the inner membrane.
Fig. 1.
A HI0853-disrupted H. influenzae Rd strain is unable to use GSSG as the sole source of sulfur. Wild-type and HI0853::Cm strains either transformed with plasmid pACYC853 (containing HI0853 with promoter and terminator regions) or empty vector (pACYC184) were streaked on cdMIc plates supplemented with either 50 μM cystine (A) or 50 μM GSSG (B). Plates were incubated for 20 h at 37 °C and photographed.
The identification of HI0853 as an essential component of the GSSG uptake pathway is a most surprising finding. This is because for nearly two decades this same ORF has been studied as the periplasmic heme-binding lipoprotein, HbpA (26–29). HbpA was originally identified and annotated using a H. influenzae subgenomic library screen conferring hemin-binding characteristics to the E. coli recipient host (26).
To resolve the intriguing ligand preferences of HbpA and its emerging role in glutathione transport, we conducted a series of experiments in vivo using whole cells (growth and competition assays of wild-type Rd versus hbpA::Cm), and in vitro employing purified recombinant HbpA. To avoid confusion, the product of HI0853 we have linked to GSSG uptake will hereafter be referred to as HbpA.
Cellular Growth Assays Point to HbpA Specificity for GSSG, GSH, and Hemin.
Strain hbpA::Cm and wild-type H. influenzae Rd displayed comparable growth curves in cdMIc medium supplemented with a wide range of cysteine-containing peptides (the dipeptides Gly-Cys, Cys-Gly, and the disulfide of Cys-Ala, the tripeptides Gly-Cys-Gly, and Gly-Gly-Cys, and the tetrapeptides Gly-Gly-Gly-Cys, and Val-Thr-Cys-Gly), indicating that HbpA does not recognize ligands of the canonical dipeptide-binding protein DppA (or that efficient redundant systems are at play). Table 1 scores a number of glutathione derivatives and elementary components that either can be metabolized by H. influenzae Rd to complement its cysteine auxotrophy or can compete with the import/use of GSSG as a cysteine source. Out of the tested molecules, only GSSG, GSH, S methylglutathione, γ-glutamyl-methylthioglycyl-glycine [γ-Glu-Gly(SCH3)-Gly], and glutathione amide (GASH) emerged as candidate ligands for HbpA. Additionally, our data reveal the apparent intolerance of HbpA for (i) isosteric replacements of the glutathione backbone carboxy terminus with small functional groups (e.g., the amidation of the glycine carboxylate decimates binding), and (ii) for replacement of the cysteine sulfur with increasingly bulkier groups (e.g., S methylglutahione inhibits GSSG uptake, but S propylglutathione does not).
Table 1.
Cell growth assays to probe HbpA-dependent ligand import in H. influenzae Rd
Columns A and B: Growth of wild-type (column A) or hbpA::Cm cells (column B) in the presence of the indicated glutathione derivatives (at 50 μM) as sole sources of sulfur. Growth of wild-type cells and lack of growth for hbpA mutants identifies the molecule as a potential cargo for the HbpA-dependent glutathione importer. Column C: Scoring of the ability of the indicated glutathione derivatives (at 3 mM) to inhibit growth of wild-type cells in cdMIc-medium supplemented by 50 μM GSSG. Inhibition of growth possibly indicates a direct interaction of the tested molecule with the HbpA-dependent glutathione importer
*Growth of H. influenzae Rd culture was measured spectrophotometrically in cdMIc-medium supplemented with 50 μM of the candidate ligands. A plus sign and a minus sign indicate “growth” and “no growth,” respectively.
†
Growth of hbpA::Cm culture was measured spectrophotometrically in cdMIc-medium supplemented with 50 μM of the candidate ligands. A plus sign and a minus sign indicate “growth” and “no growth,” respectively.
‡
Growth of H. influenzae Rd inoculated in GSSG (50 μM)-supplemented cdMIc-medium, in the presence (3 mM) and absence of ligands tested. A minus sign indicates no differences in growth characteristics, while a plus sign indicates inhibition of growth in the ligand-supplemented culture. Both positively scored derivatives at 3 mM inhibit growth completely and are nontoxic in growth experiments using cystine-supplemented cdMIc medium.
§
DTT (250 μM) was included in duplicated samples to rule out inconsistencies due to intermolecular disulfide formation and to keep the peptides reduced during the course of the experiments. DTT becomes toxic at concentrations higher that 0.5 mM.
∥
Growth is barely detected at 50 μM. However, GASH at 500 μM or higher could establish GSH-dependent growth characteristics of wild-type cells.
When compared to wild-type H. influenzae Rd, aerobic growth of the hbpA::Cm mutant as well as the HI1187 (dppB) transposon mutant in cystine-supplemented cdMIc medium containing limiting levels of hemin is characterized by a delayed onset of growth and longer doubling times (Fig. S2). This behavior suggests a redundant role of hbpA and its cognate permease in hemin transport and is consistent with a number of independently derived hbpA mutants in serotype b and nontypeable serotype backgrounds reported previously (27–29).
HbpA binds GSSG and GSH with Physiologically Relevant Affinity.
To probe the substrate specificity further, we employed two complementary lines of experimentation using recombinant HbpA. In the first instance, we used thermal denaturation assays to screen for putative ligands, followed by isothermal titration calorimetry to quantify candidate interactions.
Thermal denaturation assays offer a convenient initial screening for potential binders, as ligand binding to the native state of a protein (in the absence of binding to the denatured state) will lead to an increase in the melting temperature (Tm). Here, we applied a fluorescence-based thermal shift assay, the Thermofluor assay, employing the fluorescent dye Sypro orange that preferentially binds to the unfolded state of proteins. Fig. S3 shows the temperature-induced changes in relative fluorescence of 100 μg HbpA as a function of candidate ligands at 1 mM. We have found that only GASH, S-Me-GSH, GSH, and GSSG significantly affected the transition midpoint temperature of the apo form (Fig. S3), indicating that they may indeed bind to HbpA, as was already suggested by our cell growth assays (Table 1). Importantly, these experiments provided a first approximation of the ligand-binding preferences of HbpA with GASH being the weakest binder and GSSG the strongest.
Isothermal titration calorimetry (ITC) was subsequently used to determine the equilibrium dissociation constants (at 37 °C) for the interaction of HbpA with GSSG, GSH, and S-me-GSH. A typical ITC trace, showing the raw and integrated data for the interaction with GSSG, is shown in Fig. 2A. This binding isotherm could be fitted readily to a 1∶1 binding stoichiometry (n = 1.01 ± 0.01) to yield a Kd of 12.9 ± 0.3 μM and a highly endothermic apparent ΔH° of 86.7 kJ/mol. Consequently, HbpA/GSSG complex formation is entropically driven and is characterized by 115.7 kJ/mol of apparent TΔS°. For the S-Me-GSH titration (Fig. S4c), we obtained ΔH°, TΔS°, and Kd values of 14.3 kJ/mol, 36.1 kJ/mol, and 212 ± 17 μM, respectively.
Fig. 2.
Characterization of ligand binding to HbpA. (A) ITC characterization of the interaction of HbpA with GSSG. (Upper) Represents the raw data; (Lower) illustrates the modeled data and the corresponding thermodynamic profiles. ITC thermograms for the interaction of HbpA with GSH and S-me-GSH are presented in Fig. S4. (B) Absorption spectrum of 25 μM HbpA and 40 μM hemin corrected by 40 μM hemin in the reference cell. The hemin 385 nm Soret peak is shifted to 415 nm as a result of its interaction with HbpA. (C) Native-PAGE analysis of 10 μg HbpA titrated with 0.0 mM hemin (lane 1), 0.1 mM hemin (lane 2), 0.5 mM hemin (lane 3), 1.0 mM hemin (lane 4), 2.0 mM hemin (lane 5), and 3.0 mM hemin (lane 6). The disappearance of apo-HbpA was densitometrically quantified and plotted against hemin concentration (red squares). A Kd of 655 ± 109 μM was calculated for the HbpA-hemin interaction upon fitting the data to a simple binding isotherm equation using a nonlinear least-square fitting routine (green curve). (D) Schematic summary of the presently identified HbpA ligands according to affinity.
Because no distinct enthalpy changes were observed upon titrating GSH, we opted for an indirect determination of Kd by displacement ITC. Hence, two additional ITC experiments were performed. In the first experiment, GSSG was titrated into the HbpA-containing sample cell containing 100 μM GSH (Fig. S4a), whereas in the second GSSG was titrated against HbpA in the presence of 400 μM GSH (Fig. S4b). Inclusion of GSH in the isothermal cell shifted the Kd,GSSG (12.9 μM) to apparent values of 34.7 μM and 103 μM, respectively, without significantly affecting the apparent ΔH° values. These shifts correspond to a competitive inhibition constant of 56.4 ± 3.0 μM for the enthalpically neutral interaction of GSH with HbpA.
Although we were able to successfully apply such a dual experimental approach to characterize ligand binding to HbpA for most candidate ligands, efforts to measure hemin binding in a similar fashion proved to be inadequate. For instance, hemin is inherently unsuitable for our thermal denaturation assays due to its overlapping absorbance maximum with the fluorescence behavior of the Sypro orange dye used as fluorescent agent. Moreover, ITC titrations of HbpA (13 μM) with hemin resulted in thermograms that were indistinguishable from control titrations, which suggested a low affinity complex or possibly a zero enthalphy-change interaction. We therefore sought to employ alternative experimental approaches to probe the HbpA-hemin interaction.
HbpA Binds Hemin with Low Affinity.
A first demonstration that HbpA binds hemin came from UV-Vis absorption spectroscopy performed on recombinant HbpA. The absorption spectrum of hemin features a peak in the Soret band region at about 385 nm. Upon mixing the hemin with HbpA a red shift of this Soret band to 415 nm could be observed (Fig. 2B), suggesting HbpA-mediated perturbation of the electronic structure of the hemin iron. Titration of HbpA with increasing concentrations of hemin up to 200 μM, the upper detection limit of the spectrophotometric assay, resulted in a linear increase of the difference absorption at 415 nm without reaching a maximum. This indicated that although HbpA recognizes hemin, it does so with low affinity in the high sub-mM range.
To obtain a more accurate affinity estimate, we studied the interaction between HbpA and hemin by native PAGE, a method that has previously been successfully employed to study the interaction of heme-binding proteins with heme (30, 31). We observed that hemin-loaded HbpA, as judged by visual inspection (red-brownish bands) and heme staining with 2,3′, 5, 5′-tetramethylbenzidine (TMBZ)/H2O2, migrated faster in native PAGE when compared to the apo form. To circumvent the inherent experimental limitations of densitometry on very intense heme stains, we relied on densitometric scanning of Coomassie blue-stained gels to determine the relative amounts of apo- and hemin-loaded HbpA as a function of increasing hemin concentrations (Fig. 2C). By plotting the optical densities of bands on the gel, and fitting them to a single binding isotherm (Fig. 2C), a Kd of 655 ± 109 μM was calculated for the HbpA-hemin interaction.
Taken together, our diverse binding studies provide a first quantitative overview of the substrate preferences of HbpA (Fig. 2D).
Crystal Structure of HbpA in Complex with GSSG.
We pursued structural characterization of HbpA and close homologues thereof in order to reveal the structural basis for glutathione binding by HbpA-family proteins. We were able to determine the crystal structure of HbpA from Haemophilus parasuis (Hp_HbpA), in complex with GSSG to 1.85-Å resolution (Table S1). Hp_HbpA is very similar to H. influenzae HbpA in a number of ways: (i) both proteins are encoded by stand-alone genes that are not part of the operon coding for the cognate dipeptide permeases, (ii) the two proteins share 74% sequence identity, and (iii) both proteins have a very similar ligand-binding profile as determined by thermal denaturation assays (Fig. S3c). Interestingly, Hp_HbpA does not bind hemin at all in our native PAGE gel shift assays. Hp_HbpA adopts the pear-shaped, two-domain α/β-fold characteristic for bacterial dipeptide- and oligopeptide-binding proteins such as E. coli DppA (32), Salmonella typhimurium OppA (33), Bacillus subtilis AppA (34), and Lactococcus lactis OppA (35) (Fig. 3A). GSSG is bound at full occupancy at a large solvent-filled interface between the N-terminal and C-terminal halves of Hp_HbpA (Fig. 3B and Fig. S5). The two glutathione legs (designated as GS-I and G-II) are bound asymmetrically in two well-defined compartments, and each make numerous specific interactions with both the N- and C-terminal domains of Hp_HbpA and ordered solvent molecules (Table S2).
Fig. 3.
Structure of HbpA from H. parasuis in complex with GSSG. (A) Ribbon diagram showing an overlay of unliganded Dpp (PDB ID code 1DPE) with HbpA from H. parasuis. The structures were superposed with respect to their N-terminal domains. (B) Binding of GSSG to the HbpA interdomain interface. The two glutathione legs are labeled as GS-I and GS-II. For clarity some interactions have been omitted. A listing of all possible interactions is presented in Table S2. The figure was created with PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC).
Discussion
The molecular mechanisms dedicated to the transport of glutathione, a pleiotropic and often vital redox molecule across all branches of life, have remained largely uncharacterized despite its outspoken biological importance. The fact that glutathione-related cargos can be in the oxidized, reduced, or S-conjugated forms of glutathione depending on the cellular context and direction of transport (import or export) implies a well-defined modulation of both the priming and specificity of membrane-associated glutathione transporters. Here, we exploited the auxotrophy of the bacterial human pathogen H. influenzae for glutathione (8) to identify and characterize at the molecular and structural level possible glutathione transport mechanisms in bacteria. We have now shown that glutathione import in H. influenzae is mediated by the dipeptide permease DppBCDF and relies on the glutathione-binding properties of its cognate PBP, which we have identified as the periplasmic lipoprotein HbpA. The four Dpp permease components (the membrane-spanning domains DppB and DppC, and the nucleotide-binding domains DppD and DppF) are encoded by a single operon (HI1184-HI1187), whereas the dedicated PBP is the product of a stand-alone ORF (HI0853).
Arguably, the most striking result of our work is that it is the well-studied HbpA, a protein hitherto considered to be an important player in heme uptake (26–29), that primes the Dpp permease for glutathione import by specifically binding oxidized and reduced forms of glutathione. It has long been recognized that HbpA shares close homology with bacterial dpp-like proteins (typically > 50% sequence identity), which led to proposals about how it could serve as a binding platform for heme (36). Although HbpA has been implicated in heme acquisition, its affinity for heme was never quantified in order to place the role of HbpA in a physiological context. Earlier studies focusing on the binding of hemin-agarose to native triton-solubilized HbpA had already suggested that even though native triton-solubilized HbpA binds hemin-agarose, it does so very weakly (26). Our quantitative determination of a low affinity for hemin by HbpA (Kd = 655 μM) is consistent with those findings. When one takes into account that PBPs shown to be important for heme uptake display Kd values for hemin of 50 μM or better (30, 37) and that HbpA from H. parasuis does not bind hemin at all, our results strongly suggest that the main physiological role of HbpA does not lie in heme acquisition. On the other hand, the growth phenotypes reported here and by other investigators for HbpA-deficient strains tend to be proportional to the available hemin and cancel under anaerobic growth conditions (27–29). In addition, a recent study using a hbpA::Sp insertion mutant concluded that HbpA is redundantly involved in hemin utilization by H. influenzae (27). As heme is believed to remain well below mM concentrations in the periplasm (38), other yet unidentified mechanisms would have to be employed to enhance the in vivo affinity of HbpA for heme. For instance, this could be an activating interaction with the Dpp permease or the involvement of accessory protein(s). Nonetheless, given that hbpA mutants are viable under aerobic conditions, the debate of whether HbpA is physiologically relevant for heme acquisition by H. influenzae will likely be resolved only when compensatory systems are identified.
Our binding studies aiming to probe the ligand repertoire of HbpA in more detail have shown unequivocally that HbpA binds oxidized and reduced glutathione with physiologically relevant affinities [Kd (GSSG) = 12.9 μM and Kd (GSH) = 56.4 μM] (Fig. 2A and D and Fig. S4). This is not what one would have predicted given that HbpA is 54% identical to E. coli DppA, a PBP known to bind di- and tripeptides (39, 40), heme (30), and the heme precursor δ-aminolevulinic acid (δ-ALA) (41). Indeed, many dpp-like proteins exhibit significant ligand-binding promiscuity in terms of peptide length and side-chain content (35). Considering that lung epithelial lining fluid in humans contains approximately 415 μM GSH and 20 μM GSSG (42), the herein reported affinities of HbpA for GSSG and GSH are certainly high enough to reflect those of a dedicated glutathione PBP. Moreover, these values are similar to those determined for the glutathione importer of Streptococcus mutans (Km of 18 μM) (7), and the yeast OPT family glutathione transporters Hgt1p and Pgt1/SpOPT (17–19), indicating that dedicated glutathione transporters could be expected to exhibit low μM affinities for their cargo. Interestingly, the only modification tolerated by HbpA on the glutathione backbone is the methylation of the cysteine sulfur. We propose that this may reflect a mechanism for excluding potentially toxic glutathione conjugates from the import process, while sequestering useful forms of glutathione, i.e., those that could serve as a source of cysteine or could function as cofactors and redox modulators, for import. Additional discussion of the possible role of HbpA in virulence is provided in SI Text.
The first snapshot of a physiologically relevant complex of an HbpA-family protein with its ligand cargo has provided the missing link for understanding at the atomic level how HbpA-family proteins could serve as glutathione-binding platforms for priming their cognate dipeptide permeases (Fig. 3). In its GSSG-bound form H. parasuis HbpA adopts a collapsed conformation about the hinge region connecting the N- and C-terminal domains, to sandwich a single GSSG molecule. This is consistent with the general mode of action of bacterial periplasmic binding proteins, whereby substrate binding between the N- and C-terminal domains shifts the equilibrium toward a closed state, in a process often referred to as a Venus flytrap mechanism. In their closed, ligand-bound forms PBPs associate with their cognate membrane-embedded pore to deliver the cargo for translocation (21, 43). Interestingly, the asymmetric binding of GSSG in Hp_HbpA whereby GS-I and GS-II adopt a compact versus extended conformation, respectively (Fig. 3B), is similar to that adopted in glutathione reductase, the only other structurally characterized protein known to bind GSSG (44). In that case, however, the relative orientation of GS-I and GS-II to each other is drastically different. Attempts to model hemin into the observed ligand-binding site of Hp_HbpA led to the conclusion that this binding site is grossly incompatible with such ligand. If HbpA does harbor a specific binding site for its weak affinity for hemin, it would have to be elsewhere on its scaffold. In this regard, there is precedence for nonoverlapping ligand-binding sites in PBPs, as evidenced by the accommodation of distinct binding sites for heme and nickel by E. coli NikA (38). We have, in fact, investigated whether hemin and glutathione might compete for the same binding site, by repeating the absorption spectrum and native PAGE analysis in the presence of 1 mM GSSG. We found that neither the observed shift of the hemin Soret peak nor the electrophoretic mobility of HbpA in native PAGE was affected, thus ruling out the possibility for a common binding site.
The amino acids involved in numerous specific interactions between GSSG and Hp_HbpA (Table S2) are conserved in HbpA and can be traced to well-conserved sequence fingerprints across the entire HbpA family of proteins (Fig. S6). Such sequence features could be used to distinguish HbpA-like proteins with glutathione-binding properties from other homologues of the Dpp superfamily. For instance, we note the presence of a basic residue (Arg or Lys) at position 33 making electrostatic interactions with the C terminus of Gly-II in GS-II, the C-terminal glutamine at position 516 interacting with the γ-Glu residue of GS-II, and the employment of a nonproline residue at position 380 thus freeing up the peptide nitrogen for hydrogen bonding with the carbonyl oxygen of Gly-I of CS-I (Table S2). Also noteworthy is the employment of a tyrosine at position 521 that appears to serve as an aromatic bed against the disulfide of GSSG. Finally, we highlight the remarkable multivalency of Arg 379, which is the only residue interacting with both GS-I and GS-II (Fig. 3B). An arginine at this position is conserved throughout the Dpp superfamily and is typically associated with stabilizing the carboxy-terminal groups of bound peptides. In HbpA this basic role is retained but also expanded to accommodate both glutathione legs. Taken together, the structural features of GSSG binding by HbpA-family proteins highlight the versatility of the dpp fold whereby a handful of key mutations on either side of the binding interface has led to a gain of function. This notion is further supported by a phylogenetic analysis of Dpp-like proteins including the HbpA-like homologs, which clearly shows that HbpA-like proteins branch distinctly away from the remaining Dpp-like sequences (Fig. S7).
We have found that HbpA-like sequences are restricted to the Pasteurellaceae family, which are commensal Gram-negative γ-proteobacteria often associated with disease in humans and other mammals. Intriguingly, Pasteurellaceae strains that do not encode for a HbpA homologue are also devoid of a canonical dppABCDF operon, suggesting that HbpA-like proteins are obligatorily coupled to cognate dipeptide permeases and that the Dpp permease mainly functions as a glutathione pore in Pasteurellaceae. The only other prokaryotic operon known to be associated with glutathione import, the E. coli operon yliABCD, also codes for an ABC transporter (10). YliB, the system’s PBP that would be analogous to HbpA in H. influenzae, shares 27% sequence identity with HbpA and both proteins have a bacterial extracellular solute-binding protein family 5 signature.
Our combined functional and structural studies have led to the unmasking of the true physiological role of HbpA-family proteins as PBPs that prime glutathione import through the dipeptide permeases in the Pasteurellaceae family of bacteria. The uncovering of the major physiological function for HbpA illustrates that even in seemingly well-characterized protein families, there are functional aspects yet to be discovered. These findings also prompt us to propose a name change for HbpA to Glutathione-Binding Protein A, or GbpA in short. Besides the obvious need to rename the protein to reflect its biological function accurately, the new name can facilitate a more unambiguous functional annotation of bacterial genomes with respect to GbpA-like proteins.
Methods
A detailed account of all experimental procedures is given in SI Text.
Data Availability
Data deposition: The atomic coordinates and crystallographic structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3M8U).
Acknowledgments.
We thank the Swiss Light Source (Villigen, Switzerland) and the staff of beamline X06DA for synchrotron beamtime allocation and technical support. This work was supported by Grant 3G020506 to B.V. and B.D. and Grant 3G064307 to S.N.S. via the Research Foundation Flanders, Belgium (FWO). B.V. and J.E. are postdoctoral and predoctoral research fellows of the FWO, respectively.
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Data deposition: The atomic coordinates and crystallographic structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3M8U).
We thank the Swiss Light Source (Villigen, Switzerland) and the staff of beamline X06DA for synchrotron beamtime allocation and technical support. This work was supported by Grant 3G020506 to B.V. and B.D. and Grant 3G064307 to S.N.S. via the Research Foundation Flanders, Belgium (FWO). B.V. and J.E. are postdoctoral and predoctoral research fellows of the FWO, respectively.
Notes
*This Direct Submission article had a prearranged editor.
Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium
Author contributions: B.V. and S.N.S. designed research; B.V., J.E., A.D., and S.N.S. performed research; B.D. contributed new reagents/analytic tools; B.V., J.E., and S.N.S. analyzed data; and B.V. and S.N.S. wrote the paper.
Competing Interests
The authors declare no conflict of interest.
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B. Vergauwen,
J. Elegheert,
A. Dansercoer,
B. Devreese,
& S.N. Savvides,
Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA, Proc. Natl. Acad. Sci. U.S.A.107 (30) 13270-13275,https://doi.org/10.1073/pnas.1005198107(2010).
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