title: Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA.
abstract: 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.
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium. bjorn.vergauwen@ugent.be
authors.forename: Bjorn
authors.initials: B
authors.lastname: Vergauwen
authors.forename: Jonathan
authors.initials: J
authors.lastname: Elegheert
authors.forename: Ann
authors.initials: A
authors.lastname: Dansercoer
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Savvas N
authors.initials: SN
authors.lastname: Savvides
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000737:chemistry / Q000378:metabolism; D006193:Haemophilus influenzae / Q000235:genetics / Q000378:metabolism*; D006427:Hemin / Q000737:chemistry / Q000378:metabolism; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C097952:HbpA protein, Haemophilus influenzae; D008074:Lipoproteins; D033902:Periplasmic Binding Proteins; D006427:Hemin; D005978:Glutathione; D019803:Glutathione Disulfide