Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins
© Vergauwen et al; licensee BioMed Central Ltd. 2011
Received: 12 September 2011
Accepted: 16 November 2011
Published: 16 November 2011
The Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires the redox-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-studied dipeptide (Dpp) permease of Escherichia coli. The solute binding protein (SBP) that mediates glutathione transport in H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family 5 SBP's. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBP was 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 role for GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought to characterize 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.
Lipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificity was evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin was observed for all five GbpA homologous test proteins. Three out of five test proteins were found to bind glutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of the tested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequence alignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separate phylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determined the crystal structure of one of the GbpA family outliers from H. parasuis. Comparisons thereof with the previously determined structure of GbpA in complex with oxidized glutathione reveals the structural basis for the lack of allocrite binding capacity, thereby explaining the outlier behavior.
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 studies strongly implicate GbpA family SBPs in the priming step of ABC-transporter-mediated translocation of useful forms of glutathione across the inner membrane, and rule out a general role for GbpA proteins in heme acquisition.
ATP-binding cassette (ABC)-transporters exist in all three kingdoms of life and transport a large variety of substrates across biological membranes. In addition to their well-documented role in solute transport, a diversity of sensory functions have been assigned that implicate ABC-transporters in the maintenance of cell integrity, responses to environmental stresses, cell-to-cell communication and cell differentiation and in pathogenicity. Based on the direction of transport, ABC transporters can be classified as either exporters or importers. Both classes are characterized by the coupling of two nucleotide-binding domains (NBD) and two transmembrane domains (TMD). In the case of ABC importers, which are found exclusively in prokaryotes, a fifth domain, termed the solute binding protein (SBP), is part of the functional unit . SBPs bind their ligands with high affinity and deliver them to the permease unit (the TMDs), where the substrate is released into the translocation pore upon ATP binding and hydrolysis in the NBDs [2, 3]. SBPs are located in the periplasm of Gram-negative bacteria, or lipid-anchored to the cell wall, or fused to the TMD in the case of Gram-positive bacteria and Archaea . Although SBPs of Gram-negative bacteria exist predominantly as stand-alone periplasmic proteins, they are sometimes connected in a fusion protein with the TMD  or observed lipid-anchored to the inner membrane [5, 6]. The physiological relevance of the immobilized versions of SBPs remains largely unaddressed in the literature.
Based on sequence homology analyses, the bacterial SBP superfamily has been classified into 8 clusters, with cluster 5 comprising dipeptide binders (DppA family), oligopeptide binders (OppA family) and nickel specific SBP's (NikA family) . Continuous family updates by the Transporter Classification Database http://www.tcdb.org has now led to a cluster 5 SBPs containing up to 27 different subfamilies that are associated with translocation cargos as diverse as - in addition to di-and oligo-α-peptides and nickel substrates - antimicrobial peptides, δ-aminolevulinic acid, heme, plant opines, carbohydrates, the osmoprotective proline betaine, and the metal-chelater ethylene diamine tetraacetate. The most recent addition to the SBP family is termed GbpA (TCID: 3.A.1.5.27), a lipoprotein from Haemophilus influenzae, which binds reduced (GSH) and oxidized (GSSG) forms of glutathione to prime the dipeptide-DppBCDF ABC-transporter for glutathione translocation across the inner membrane . Structural studies of the highly homologous GbpA from H. parasuis in complex with GSSG have revealed structural features that typify cluster 5 SBPs, namely, a pear-shaped, two-domain α/β-fold that collapses about the hinge region connecting the N-and C-terminal domains to sandwich the molecular cargo, in this case a single GSSG molecule . In the absence of ligand, SBP's are flexible with the two domains rotating around the hinge and existing largely in the open conformation with both domains separated. Substrate binding induces the closed conformation, and the ligand is trapped at the interface between the two domains, according to what has been termed the "Venus Fly-trap" mechanism . The structural analysis of GbpA in complex with GSSG has identified many specific interactions between GSSG and its cognate SBP that may be helpful in the delineation of the entire GbpA family . The discovery that GbpA mediates glutathione transport in H. influenza came as a complete surprise as this protein was previously thought to be a heme-binding protein, accordingly annotated HbpA, and was implicated as a binding-platform for heme [5, 10]. Nonetheless, GbpA does bind hemin, albeit weakly with an apparent Kd of 655 μM , and a possible role for GbpA and DppBCDF in heme acquisition has been described [10, 11]. In this regard, GbpA presents itself as a good example of the high degree of substrate promiscuity especially common among cluster 5 SBPs [12–15].
In light of our recent report on the functional reannotation of HbpA to GbpA , the present study was designed to elucidate further and refine this emerging SBP subfamily of glutathione-binding proteins and to clarify the roles of such proteins in glutathione and heme acquisition. GbpA homologs were identified employing BLAST and their clustering in the novel GbpA family was established based on structure-based motif fingerprinting. To ascertain the GbpA family functionally, we subsequently explored the ligand preferences of five representative GbpA homologous proteins. As the GbpA from H. influenzae is lipidated in vivo, we also incorporated in our test protein set GbpA homologous sequences that were not preceded by a peptidase II modifiable leader peptide, thereby providing the opportunity to uncover lipidation-dependent functional effects. Our studies indicate that GbpA family members are exclusively found in the Gram-negative Pasteurellaceae, where they have evolved by gene duplication from a canonical DppA sequence to prime the transport of physiologically useful forms of glutathione. Our data on the other hand do not support a general role for GbpA family proteins in heme acquisition. Finally, a phylogentically distinct cluster of GbpA homologues was identified, which appears to lack binding capacity not only for glutathione and other peptide ligands, but heme as well, thus casting a new twist in the possible substrate preferences of GbpA-like proteins.
Results and Discussion
Interestingly, the HbpA2 clade diverged significantly from both the GbpA and DppA signature sequences. In fact, some of the strictly conserved residues that contact the ligand's charged N- and C-termini in either the GbpA or the DppA family are replaced by physicochemically dissimilar residues in the HbpA2 sequences thereby virtually disrupting critical ligand-stabilizing salt bridges (In case of GbpA-GSSG binding, Arg33 substituted by a Thr, and Asp432 substituted by an Arg; in case of DppA-dipeptide binding, Asp408 substituted by an Arg). The ligand specificity of the HbpA2 clade is therefore difficult to predict, but it is highly unlikely that glutathione or dipeptides are the natural molecular cargos. Given the auxotrophic nature of Pasteurellaceae for heme and the fact that the dpp-architecture is a proven hemin-binding scaffold (E. coli DppA binds hemin with a 10 μM affinity  and also GbpAHi displays an, albeit low, affinity for hemin ) it is tempting to speculate that HbpA2 proteins may play a role in heme transport.
To document the heme-binding characteristics of the GbpA family, to verify the role of the posttranslational 1,2-diacylglycerol-modification of GbpA proteins in terms of glutathione and heme binding, and to establish the ligand-preferences of the HbpA2 family, we selected in addition to GbpAHi yet another GbpA lipoprotein (from A. pleuropneumoniae, GbpAAp), 2 non-lipoprotein GbpA's (GbpAHp and GbpAPm from H. parasuis and P. multocida, respectively), and 2 HbpA2 proteins (HbpA2Hp and HBPA2Ap from H. parasuis and A. pleuropneumoniae, respectively), for further study.
Summary of results obtained from thermal shift assays for the identification of GbpA- and HbpA2-family ligands out of a test set of typically family 5 SBP allocrites.
T m (°C) a
glutathione cysteine disulfide
Summary of the dissociation constants for the interaction of our GbpA-family test set with the physiologically relevant glutathione forms (GSSG and GSH), and the artificial S-methylglutathione (S-me-GSH) as determined by ITC at 37°C.
12.9 ± 0.3
0.33 ± 0.05
2.1 ± 0.2
0.15 ± 0.03
56.4 ± 3.0
1.58 ± 0.2
1.9 ± 0.1
0.26 ± 0.05
212 ± 17
1.17 ± 0.05
0.90 ± 0.07
0.55 ± 0.04
We here have provided a biochemical and phylogenetic delineation of the GbpA-family of glutathione-binding proteins. We showed that the GbpA proteins likely evolved exclusively within the Pasteurellaceae lineage by gene duplication from an already present dipeptide-binding protein, DppA, thereby explaining our previously reported functional annotation of GbpA proteins as periplasmic binding proteins that prime glutathione import to the cytoplasm via the cognate Dpp-ABC transporter . GbpA proteins are specific for the glutathione backbone, but can tolerate S-modifications to different extends. This slightly promiscuous behavior probably resulted from the evolutionary tailoring of the GbpA scaffold to also accommodate useful disulfides of glutathione, such as GSSG and glutathione cysteine disulfide. Although GbpA proteins were formerly known as heme-binding proteins, an important implication of our work concerns the awareness that they clearly do not have a general role in heme acquisition. Apart from GbpA and/or DppA representatives, some Pasteurellaceae also carry the genetic information for a close, although phylogenetically distinct homolog, which we have termed HbpA2 in the present paper. Because we were unable to identify a molecular interaction partner for these paralogous HbpA2 proteins, their in vivo role will have to await further study. In any case, the current annotation as "heme-binding protein or HbpA" for HbpA2-family members is clearly inaccurate and databases should be rectified accordingly (e.g family 5 SBP with no known function).
Wild-type strain H. influenzae Rd was purchased from the American Type Culture Collection (Manassas, Va.). The P. multocida and A. pleuropneumoniae clinical isolates used in this study were a kind gift of Dr. Mario Vaneechoutte (Deptartment of Clinical Chemistry, Microbiology, and Immunology, University Hospital, Ghent, Belgium). The H. parasuis strain used in this study was isolated from the nasal cavity of a clinically healthy pig and was kindly provided by Dr. Filip Boyen (Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium).
Production and purification of recombinantSBP's
The construction of the expression plasmids pET-GbpAHi and pET-GbpAHp is described in ref. . The remaining proteins in this study were overexpressed using similarly constructed plasmids also based on the pET20b vector template. The leader peptides of the respective proteins were predicted using the SignalP 3.0 server http://www.cbs.dtu.dk/services/SignalP/ and the LipoP 1.0 server http://www.cbs.dtu.dk/services/LipoP/ and replaced by the PelB leader peptide provided by the pET20b plasmid. In case of the A. pleuropneumoniae gbpA gene sequence, the codon that translates to the N-terminal Cys was furthermore replaced by the Met codon. Therefore the mature proteins started at positions 23, and 22 for the P. multocida, and A. pleuropneumoniae GbpA family members, respectively, and at positions 19, and 21 for the HbpA2 SBP's of H. parasuis, and A. pleuropneumoniae, respectively. All test protein coding sequences were extended with a his-tag to facilitate purification. The respective genes were PCR-amplified using forward and reverse primers (5' to 3'), respectively - with the cloning (restriction) site underlined and identified between brackets: GbpAPm (CCATGG ATAATAAAACCTTTATTAACTGC [Nco I], GCGGCCGC ATCCGCTAACTTAGTGC [Not I]); GbpAAp (CCATGG ATGATAAAAATGCGGACG [Nco I], GCGGCCGC GTCGGCTAATTTTGTACCG [Not I]); HbpA2Hp (GATATC TCGGCACCGACAAATACATTG [Eco RV], CTCGAG TTAAGGCTTCAGACTTACGCCAT [Xho I]); HbpA2Ap (CCATGG CAGCGCCGGCACATACTTTAG [Nco I], GCGGCCGC TTCCGTTAGACTCACATTATAG [Not I]).
The proteins were expressed in E. coli and purified using a three-step chromatographic protocol (IMAC, followed by anion-exchange, and size-exclusion chromatography) as described in ref. . The concentration of purified proteins was determined by the Bio-Rad Protein Assay with bovine serum albumin as the standard.
Native PAGE heme-binding gel shift assay
Hemin stock solutions were prepared by dissolving bovine hemin chloride (Sigma-Aldrich) in 100 mM NaOH prior to 10-fold dilution in double distilled water. These solutions were then neutralized to pH 7.5 using HCl and filtered through a Millex-GP 0.22 μm filter unit (Millipore). Stock concentrations were determined spectrophotometrically (ε385 = 58,400 M-1 cm-1), and the solutions were used within a day after preparation. Purified test protein (10 μg) was incubated with 0.5 mM hemin or water alone for 45 min at room temperature and subjected to native PAGE as described previously . Hemin-complexed bands were visualized in-gel by their intrinsic peroxidase activity using 2,3',5,5'-tetramethylbenzidine and H2O2. The hemin-complexed protein species migrated faster compared to the apo-forms as was already described for other hemin-binding proteins [8, 19, 24].
Thermal denaturation assays
Thermofluor thermal shift assays were conducted in a C1000 thermal cycler equipped with a CFX96 optical reaction module (Bio-Rad). The microplate wells were loaded with 25-μL solutions, containing 100 μg test protein, 2 × Sypro orange (Molecular Probes), and 1 mM of the test chemicals in 10 mM Tris-HCl, pH 8.0. The plates were sealed with Microseal B film (Bio-Rad) and heated from 30°C to 90°C at a rate of 2°C per min. The unfolding reactions were followed by simultaneously monitoring the relative fluorescence (FRET settings) using the charge-coupled device camera. The inflection point of the fluorescence versus-temperature curves was identified by plotting the first derivative over the temperature, and the minima were referred to as the melting temperatures (Tm).
Isothermal titration calorimetry (ITC)
Experiments were carried out using a VP-ITC MicroCalorimeter (MicroCal) at 37°C, and data were analyzed using the Origen ITC analysis software package supplied by MicroCal. Purified test proteins were dialyzed overnight against 10 mM Tris-HCl, pH 7.4, at 4°C. The resultant dialysis buffer was then used to dissolve the test compounds. Protein concentrations were measured spectrophotometrically using the respective theoretical extinction coefficients at 280 nm as calculated from the mature protein sequences at http://web.expasy.org/protparam/. GSSG concentrations were determined by the absorbance change at 340 nm resulting from the glutathione reductase-catalyzed NADPH-dependent conversion of GSSG to 2GSH (ε340 = 6,200 M-1 cm-1). GSH concentrations were determined by the reaction with Ellman's reagent (ε412 = 14,000 M-1 cm-1). All solutions were degassed prior to use. Titrations were always preceded by an initial injection of 3 μL and were carried out using 10-μL injections applied 300 s apart. The sample was stirred at a speed of 400 rpm throughout. Test compounds were always injected into the HbpA-containing sample cell. The heats of dilution were negligibly small for the titration of each ligand into buffer; hence the raw data needed no correction. The thermal titration data were fit to the one binding site model to determine the dissociation constant, Kd. Several titrations were performed to evaluate reproducibility.
Crystallization and structure determination of HbpA2 from H. Parasuis
Crystallization of HbpA2Hp (10 mg/mL in 10 mMTris-HCl pH 8.0, 100 mMNaCl) was screened using a Mosquito crystallization robot (TTP LabTech) based on 200 nL crystallization droplets (100-nL protein sample and 100-nL crystallization condition) equilibrated in sitting-drop geometry over 25-μL reservoirs containing a given crystallization condition. This led to the development of already well-formed rod-shaped crystals in condition 39 of the Hampton Research Index screen (0.1 M HEPES pH 7.0,30% v/v jeffamine ED-2001). This condition was optimized using a bigger "sitting-drop" geometry as follows. Crystallization droplets consisting of 1-μL protein sample and 1 μL 0.1 M HEPES pH 7.0, 30% v/v jeffamine ED-2001, were equilibrated against 0.75-mL reservoir solution containing 5-20% wt/v saturated ammonium sulfate. Diffraction quality crystals of HbpA2Hp grew overnight as clusters of easy separable crystalline rods (measuring 0.05 × 0.05 × 0.2 mm). For data collection under cryogenic conditions (100 K), single crystals were flash cooled with the help of a nylon loop directly in liquid nitrogen after a very brief incubation (typically < 30 s) in cryoprotecting solution containing 0.1 M HEPES pH 7.0, 30% v/v jeffamine ED-2001, and 20% v/v glycerol. The structure of HbpA2 from H. parasuis was determined by maximum-likelihood molecular replacement as implemented in the program suite PHASER . The search model was prepared from the structure of H. parasuis GbpA in complex with GSSG  using the program Chainsaw , based on structure-based sequence alignments. The final search model contained alanines at all nonconserved positions and was stripped from all solvent molecules and ligand. The best solution was obtained in a combined search strategy whereby we searched for the C-terminal domain first. Inspection of electron density maps calculated with Fourier coefficients 2Fo-Fc, MR, αc, MR confirmed the solution as evidenced by extra density for missing structural elements and side chains. Model (re)building was carried out via a combination of automated methods as implemented in the PHENIX suite  and manual adjustments using the program COOT . Crystallographic refinement and structure validation was carried out using PHENIX and Buster [27, 29].
This work was supported by Grant 3G020506 to BV and Grant 3G064307 to SNS via the Research Foundation Flanders, Belgium (FWO). BV and RVDM are postdoctoral and predoctoral research fellows of the FWO, respectively. We thank the Swiss Light Source (Villigen, Switzerland) for synchrotron beamtime allocation and the staff of beamline PXIII for technical support. We express our gratitude to Annelies Van Raemdonck for excellent technical assistance.
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