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

<p>Abstract</p> <p>Background</p> <p>The Gram-negative bacterium <it>Haemophilus influenzae </it>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 <it>Escherichia coli</it>. The solute binding protein (SBP) that mediates glutathione transport in <it>H. influenzae </it>is a lipoprotein termed GbpA and is 54% identical to <it>E. coli </it>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.</p> <p>Results</p> <p>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 <it>H. parasuis</it>. 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.</p> <p>Conclusions</p> <p>Taken together, our studies provide for the first time a collective functional look on a novel, <it>Pasteurellaceae</it>-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.</p

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor a-chain (IL-7Ra) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Ra extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency

Structural basis of GM-CSF and IL-2 sequestration by the viral decoy receptor GIF.

Subversion of the host immune system by viruses is often mediated by molecular decoys that sequester host proteins pivotal to mounting effective immune responses. The widespread mammalian pathogen parapox Orf virus deploys GIF, a member of the poxvirus immune evasion superfamily, to antagonize GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-2 (interleukin-2), two pleiotropic cytokines of the mammalian immune system. However, structural and mechanistic insights into the unprecedented functional duality of GIF have remained elusive. Here we reveal that GIF employs a dimeric binding platform that sequesters two copies of its target cytokines with high affinity and slow dissociation kinetics to yield distinct complexes featuring mutually exclusive interaction footprints. We illustrate how GIF serves as a competitive decoy receptor by leveraging binding hotspots underlying the cognate receptor interactions of GM-CSF and IL-2, without sharing any structural similarity with the cytokine receptors. Our findings contribute to the tracing of novel molecular mimicry mechanisms employed by pathogenic viruses

A direct spectrophotometric gamma-glutamyltransferase inhibitor screening assay targeting the hydrolysis-only mode

gamma-Glutamyltransferase (GGT, E.C. 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione. Due to its central role in maintaining mammalian glutathione homeostasis, GGT is now believed to be a valuable drug target for a variety of life-threatening diseases, such as cancer. Unfortunately, however, effective tools for screening GGT inhibitors are still lacking. We report here the synthesis and evaluation of an alpha-phenylthio-containing glutathione peptide mimic that eliminates thiophenol upon GGT-catalyzed hydrolysis of the gamma-glutamyl peptide bond. The concurrent, real-time spectrophotometric quantification of the released thiophenol using Ellman's reagent creates a GGT assay format that is simple, robust, and highly sensitive. The versatility of the assay has been demonstrated by its application to the kinetic characterization of equine kidney GGT, and enzyme inhibition assays. The ability of the glutathione mimic to behave as an excellent donor substrate (exhibiting Michaelis Menten kinetics with a K-m of 11.3 +/- 0.5 mu M and a k(cat) of 90.1 +/- 0.8 nmol mg(-1) min(-1)), coupled to the assay's ability to study the hydrolysis-only mode of the GGT-catalyzed reaction, make Our approach amenable to high-throughput drug screening platforms

Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA

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

Acetyl-CoA is produced by the citrate synthase homology module of ATP-citrate lyase

Recent structural studies on ACLY1–4, a central enzyme for the biosynthesisof the critical metabolite acetyl-coenzyme A (CoA)5–11,provided a long-awaited framework to propose the catalytic mechanismof the enzyme1–3. ACLY holoenzymes across all kingdomsof life adopt conserved tetrameric structures of roughly 500 kDacomprising subunits featuring an N-terminal acyl-CoA synthetasehomology (ASH) module and a C-terminal citrate synthase homology(CSH) module. The catalytic itinerary of ACLY starts in theASH module where ATP-driven autophosphorylation leads to activationof citrate to citryl-phosphate. This enables the reaction withCoA to yield the high-energy intermediate citryl-CoA. Shuttling ofcitryl-CoA to the CSH module of ACLY, facilitated by flipping of thelong pantothenyl-arm of citryl-CoA to position the citryl-thioestermoiety in the CSH active site, sets the stage for the last step of thereaction, namely the retro-aldol cleavage of citryl-CoA into oxaloacetateand acetyl-CoA (Extended Data Fig. 1a,b). A study by Weiet al.12,13 claimed that both the formation and the retro-aldol cleavageof the citryl-CoA intermediate are catalyzed in the ASH moduleof ACLY instead, with the CSH module not serving a catalytic role.In light of previous publications1–4 on ACLY and new data presentedhere, we argue that this claim is incorrect and that the study byWei et al.12,13 suffers from critical issues, discussed below

Molecular and structural basis of glutathione import in Gram-positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans

Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-Ileg of GSSG is accommodated tightly via extensive interactions contributed by the N-and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine