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Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins.

Vergauwen B, Van der Meeren R, Dansercoer A, Savvides SN - BMC Biochem. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium. Bjorn.Vergauwen@ugent.be

ABSTRACT

Background: 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.

Results: 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.

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 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.

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

Mentions: The lack of conservation of consensus sequence fingerprints important for ligand-binding by GbpA- and DppA-family proteins (Figure 2) had already suggested that the HbpA2 proteins in our test set would fail to bind the glutathione- and dipeptide-types of ligands. Indeed, our thermofluor analyses showed that none of the two HbpA2 proteins under study were able to interact with any of the tested type 5 SBP superfamily allocrites (Table 1). Because of the possibility that the HbpA2 proteins would co-purify with their natural ligands, as observed for some other structurally characterized SBP's, such as e. g. the cysteine-complexed CjaA from Campylobacter jejuni [20] or the oligopeptide-binder AppA from Bacillus subtilis in complex with a nonapeptide [21], we sought to determine the crystal structure of HbpA2Hp hoping to elucidate an interaction with a possible ligand. The crystal structure of HbpA2Hp was determined to 2.0 Å resolution by maximum-likelihood molecular replacement (Figure 5; additional file 1, Table S1). The structure reveals the two-lobe α/β-fold architecture and β-strand topology typical for SBP-like proteins, and is essentially identical to that of the structurally characterized GbpA and DppA proteins. Crystallographic refinement and exhaustive examination of residual difference electron density maps failed to provide any evidence for a bound ligand to HbpA2. Moreover, the N- and C-terminal domains were opened by about 33 degrees with respect to the GSSG-bound GbpA and glycylleucine-bound DppA reported previously [8,16] (Figure 5A), again indicating we crystallized apo-HbpA2Hp. Importantly, the crystal structure of HbpA2Hp offers an explanation for its inability to bind peptide-like ligands. Figure 5B shows a structural superposition of residues of the GbpA ligand-binding site with only those corresponding residues in HbpA2Hp of which the physicochemical properties are significantly different as revealed by our sequence alignments (Figure 2). This analysis focuses on the C-terminal lobe, because it comprised the majority of the ligand-interacting residues as shown by the GbpAHp-GSSG complex [8] (13 out of 18 interactions), and due to the fact that it is believed to drive formation of the SBP-ligand-encounter complex [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 be critical for GSSG-binding by GbpAHp: the peptide-nitrogen of A380 hydrogen-bonds with the carbonyl oxygen of GlyI of one of the glutathione legs (GS-I); the D432 side chain carboxylate forms a salt bridge with the amino terminus of GS-I as well as H-bonds with the side chain hydroxyl groups of Y138 and Y521 thereby positioning these residues for favorable hydrophobic interactions, the side chain of S430 is involved in H-bonding with both the carboxylate- and amino-groups of the γ-glutamyl-moiety of GS-I [8]. The structural superposition in Figure 5B shows that S430 and D432 in the HbpA2Hp structure occupy the exact same position as the corresponding active site residues in GbpAHp. At the same time, A380 takes a slightly different position which would be expected due to the elimination of the special structural role of a proline residue in maintaining loop structure at this position. Our structural analysis offers direct evidence that the A380P, S430T, and D432R substitutions would be grossly incompatible with GSH and GSSG binding as they would abolish electrostatic, H-bonding and hydrophobic interactions contributions critical for binding of such ligands. A similar analysis, this time against E. coli DppA, shows that R415 in HbpA2Hp takes the exact same position as the active site residue D408 in E. coli DppA, a residue that makes a salt-bridge with the amino-terminus of the bound dipeptide ligand. Thus, R415 would prevent dipeptide ligand binding. Finally, we note that the inability of the HbpA2 proteins to interact with either glutathione or dipeptides is correlated by looking at the interspecies occurrence: 2 out of 3 species with HbpA2 genes also carry genes for both a GbpA and a DppA family member.


Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins.

Vergauwen B, Van der Meeren R, Dansercoer A, Savvides SN - BMC Biochem. (2011)

Structure of HbpA2 from H. parasuis. (A) Ribbon diagram showing an overlay of GSSG-complexed GbpA (PDB id. 3M8U; gray) with HbpA2 from H. parasuis (PDB id. 3TPA; blue). The structures were superposed with respect to their C-terminal domains. HbpA2 shows a conformation that opens the cleft between the N- and C-terminal domains about 30° relative to its ligand-complexed paralogous counterpart. GSSG is depicted in atom-colored sticks. (B) Key binding residues of the GbpA C-terminal domain to accommodate GSSG (shown in atom-colored gray sticks) are replaced in HbpA2 by counterparts (shown in atom-colored blue sticks) that are incompatible with binding peptide-like allocrites. Residue numbering is according to PDB id. 3M8U. Some key interactions are depicted as black dashed lines. For clarity some interactions have been omitted. The figure was created with PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3295651&req=5

Figure 5: Structure of HbpA2 from H. parasuis. (A) Ribbon diagram showing an overlay of GSSG-complexed GbpA (PDB id. 3M8U; gray) with HbpA2 from H. parasuis (PDB id. 3TPA; blue). The structures were superposed with respect to their C-terminal domains. HbpA2 shows a conformation that opens the cleft between the N- and C-terminal domains about 30° relative to its ligand-complexed paralogous counterpart. GSSG is depicted in atom-colored sticks. (B) Key binding residues of the GbpA C-terminal domain to accommodate GSSG (shown in atom-colored gray sticks) are replaced in HbpA2 by counterparts (shown in atom-colored blue sticks) that are incompatible with binding peptide-like allocrites. Residue numbering is according to PDB id. 3M8U. Some key interactions are depicted as black dashed lines. For clarity some interactions have been omitted. The figure was created with PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC).
Mentions: The lack of conservation of consensus sequence fingerprints important for ligand-binding by GbpA- and DppA-family proteins (Figure 2) had already suggested that the HbpA2 proteins in our test set would fail to bind the glutathione- and dipeptide-types of ligands. Indeed, our thermofluor analyses showed that none of the two HbpA2 proteins under study were able to interact with any of the tested type 5 SBP superfamily allocrites (Table 1). Because of the possibility that the HbpA2 proteins would co-purify with their natural ligands, as observed for some other structurally characterized SBP's, such as e. g. the cysteine-complexed CjaA from Campylobacter jejuni [20] or the oligopeptide-binder AppA from Bacillus subtilis in complex with a nonapeptide [21], we sought to determine the crystal structure of HbpA2Hp hoping to elucidate an interaction with a possible ligand. The crystal structure of HbpA2Hp was determined to 2.0 Å resolution by maximum-likelihood molecular replacement (Figure 5; additional file 1, Table S1). The structure reveals the two-lobe α/β-fold architecture and β-strand topology typical for SBP-like proteins, and is essentially identical to that of the structurally characterized GbpA and DppA proteins. Crystallographic refinement and exhaustive examination of residual difference electron density maps failed to provide any evidence for a bound ligand to HbpA2. Moreover, the N- and C-terminal domains were opened by about 33 degrees with respect to the GSSG-bound GbpA and glycylleucine-bound DppA reported previously [8,16] (Figure 5A), again indicating we crystallized apo-HbpA2Hp. Importantly, the crystal structure of HbpA2Hp offers an explanation for its inability to bind peptide-like ligands. Figure 5B shows a structural superposition of residues of the GbpA ligand-binding site with only those corresponding residues in HbpA2Hp of which the physicochemical properties are significantly different as revealed by our sequence alignments (Figure 2). This analysis focuses on the C-terminal lobe, because it comprised the majority of the ligand-interacting residues as shown by the GbpAHp-GSSG complex [8] (13 out of 18 interactions), and due to the fact that it is believed to drive formation of the SBP-ligand-encounter complex [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 be critical for GSSG-binding by GbpAHp: the peptide-nitrogen of A380 hydrogen-bonds with the carbonyl oxygen of GlyI of one of the glutathione legs (GS-I); the D432 side chain carboxylate forms a salt bridge with the amino terminus of GS-I as well as H-bonds with the side chain hydroxyl groups of Y138 and Y521 thereby positioning these residues for favorable hydrophobic interactions, the side chain of S430 is involved in H-bonding with both the carboxylate- and amino-groups of the γ-glutamyl-moiety of GS-I [8]. The structural superposition in Figure 5B shows that S430 and D432 in the HbpA2Hp structure occupy the exact same position as the corresponding active site residues in GbpAHp. At the same time, A380 takes a slightly different position which would be expected due to the elimination of the special structural role of a proline residue in maintaining loop structure at this position. Our structural analysis offers direct evidence that the A380P, S430T, and D432R substitutions would be grossly incompatible with GSH and GSSG binding as they would abolish electrostatic, H-bonding and hydrophobic interactions contributions critical for binding of such ligands. A similar analysis, this time against E. coli DppA, shows that R415 in HbpA2Hp takes the exact same position as the active site residue D408 in E. coli DppA, a residue that makes a salt-bridge with the amino-terminus of the bound dipeptide ligand. Thus, R415 would prevent dipeptide ligand binding. Finally, we note that the inability of the HbpA2 proteins to interact with either glutathione or dipeptides is correlated by looking at the interspecies occurrence: 2 out of 3 species with HbpA2 genes also carry genes for both a GbpA and a DppA family member.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium. Bjorn.Vergauwen@ugent.be

ABSTRACT

Background: 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.

Results: 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.

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 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.

Show MeSH
Related in: MedlinePlus