Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de ...novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue.
Background: Peroxiredoxin IV metabolizes endoplasmic reticulum-derived hydrogen peroxide.
Results: Peroxiredoxin IV structures reveal an unusually stable decamer and a sulfenylated intermediate in the enzymatic cycle.
Conclusion: The enzymatic cycle of peroxiredoxin IV involves destabilization of the active site followed by formation of a stable disulfide-bonded decamer.
Significance: Elucidating peroxiredoxin structures is required to understand how they function during oxidative stress.
Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas ...aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of D-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing D-rhamnose and not D-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.
Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the ...Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.
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•PICI Rpp protein promotes PICI transfer while blocking phage reproduction•Rpp forms a heterocomplex with helper phage TerS•Crystal structures of Rpp alone or complexed with TerS were determined•TerS complexed with Rpp switches specificity from the phage DNA to the PICI genome
Fillol-Salom et al. report that Gram-negative PICIs employ the Rpp protein to block helper phage reproduction. Rpp binds to helper phage TerS, and the Rpp-TerS heterocomplex enables PICIs to hijack the phage machinery for their own packaging. These findings reveal a mechanism used by Gram-negative PICIs to spread in nature.
The genomes of some purple photosynthetic bacteria contain a multigene
puc
family encoding a series of α- and β-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) ...complexes. To unravel this complexity, we generated four sets of
puc
deletion mutants in
Rhodopseudomonas palustris
, each encoding a single type of
pucBA
gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αβ-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αβ-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.
Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane ...receptors to obtain iron during infection. These receptors acquire iron either in concert with soluble iron-scavenging siderophores or through direct interaction and extraction from host proteins. Characterization of these receptors provides invaluable insight into pathogenesis. However, only a subset of virulence-related TonB-dependent receptors have been currently described. Here we report the discovery of FusA, a new class of TonB-dependent receptor, which is utilized by phytopathogenic Pectobacterium spp. to obtain iron from plant ferredoxin. Through the crystal structure of FusA we show that binding of ferredoxin occurs through specialized extracellular loops that form extensive interactions with ferredoxin. The function of FusA and the presence of homologues in clinically important pathogens suggests that small iron-containing proteins represent an iron source for bacterial pathogens.
The crystal structure at 4.8 angstrom resolution of the reaction center-light harvesting 1 (RC-LH1) core complex from Rhodopseudomonas palustris shows the reaction center surrounded by an oval LH1 ...complex that consists of 15 pairs of transmembrane helical α- and$\beta-apoproteins$and their coordinated bacteriochlorophylls. Complete closure of the RC by the LH1 is prevented by a single transmembrane helix, out of register with the array of inner LH1$\alpha-apoproteins$. This break, located next to the binding site in the reaction center for the secondary electron acceptor ubiquinone ($UQ_B$), may provide a portal through which$UQ_B$can transfer electrons to cytochrome$b/c_1$.
Phycocyanin (PC) is the principal pigment protein in the light-harvesting antenna of cyanobacteria. Here the biochemical characterization and the 1.51 Å crystal structure of PC from cyanobacterium ...Nostoc sp. WR13 (Nst-PC) is reported. The P63 crystal lattice is composed of the minimal biological entities of Nst-PC, the (αβ)3 trimeric rings. The structure has been refined to R factor 11.5% (Rfree 15.4%) using anisotropic atomic B factors. A phylogenetic study shows that the α and β chains of Nst-PC are significantly clustered in a distinct clade with Acaryochloris marina. The structure was examined to look for any significant differences between Nst-PC and PC from non-desert species. Only minor differences were found in the chromophore microenvironments. The tentative energy transfer pathways in Nst-PC were modeled based on simple structural considerations.
•The cyanobacterium Nostoc sp. WR13 is isolated from the desert Rann of Kachchh.•Crystal structure of phycocyanin purified from desert cyanobacterium is reported.•Evolution of pcA and pcB gene is shown by phylogenetic study.•Energy transfer pathway between chromophores of αβ heterodimer has been predicted.
Whooping cough (pertussis) is a highly contagious acute respiratory illness of humans caused by the Gram-negative bacterial pathogen Bordetella pertussis. The AT (autotransporter) BrkA (Bordetella ...serum-resistance killing protein A) is an important B. pertussis virulence factor that confers serum resistance and mediates adherence. In the present study, we have solved the crystal structure of the BrkA β-domain at 3 Å (1 Å=0.1 nm) resolution. Special features are a hairpin-like structure formed by the external loop L4, which is observed fortuitously sitting inside the pore of the crystallographic adjacent β-domain, and a previously undiscovered hydrophobic cavity formed by patches on loop L4 and β-strands S5 and S6. This adopts a ubiquitous structure characteristic of all AT β-domains. Mutagenesis studies have demonstrated that the hairpin-like structure and hydrophobic cavity are crucial for BrkA passenger domain (virulence effector) translocation. This structure helps in understanding the molecular mechanism of AT assembly and secretion and provides a potential target for anti-pertussis drug design.
The crystal structure of phycocyanin (
pr
-PC) isolated from
Phormidium rubidum
A09DM (
P. rubidum
) is described at a resolution of 1.17 Å. Electron density maps derived from crystallographic data ...showed many clear differences in amino acid sequences when compared with the previously obtained gene-derived sequences. The differences were found in 57 positions (30 in α-subunit and 27 in β-subunit of
pr
-PC), in which all residues except one (β145Arg) are not interacting with the three phycocyanobilin chromophores. Highly purified
pr
-PC was then sequenced by mass spectrometry (MS) using LC–MS/MS. The MS data were analyzed using two independent proteomic search engines. As a result of this analysis, complete agreement between the polypeptide sequences and the electron density maps was obtained. We attribute the difference to multiple genes in the bacterium encoding the phycocyanin apoproteins and that the gene sequencing sequenced the wrong ones. We are not implying that protein sequencing by mass spectrometry is more accurate than that of gene sequencing. The final 1.17 Å structure of
pr
-PC allows the chromophore interactions with the protein to be described with high accuracy.
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