Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa.

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein's role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.

PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation.

Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation of pel gene clusters has helped us identify an additional gene, pelX, that is present adjacent to pelABCDEFG in >100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to elucidate PelX function as Pseudomonas aeruginosa lacks a pelX homologue in its pel gene cluster. We found that P. protegens forms Pel-dependent biofilms; however, despite expression of pelX under these conditions, biofilm formation was unaffected in a ΔpelX strain. This observation led us to identify a pelX paralogue, PFL_5533, which we designate here PgnE, that appears to be functionally redundant to pelX In line with this, a ΔpelX ΔpgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1 Å resolution. The structure revealed that PelX resembles UDP-GlcNAc C4-epimerases. Using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Our results indicate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.

Structural Basis of the Interaction Between Ubiquitin Specific Protease 7 and Enhancer of Zeste Homolog 2.

USP7 is a deubiquitinase that regulates many diverse cellular processes, including tumor suppression, epigenetics, and genome stability. Several substrates, including GMPS, UHRF1, and ICP0, were shown to bear a specific KxxxK motif that interacts within the C-terminal region of USP7. We identified a similar motif in Enhancer of Zeste 2 (EZH2), the histone methyltransferase found within Polycomb Repressive Complex 2 (PRC2). PRC2 is responsible for the methylation of Histone 3 Lys27 (H3K27) leading to gene silencing. GST pull-down and coimmunoprecipitation experiments showed that USP7 interacts with EZH2. We determined the structural basis of interaction between USP7 and EZH2 and identified residues mediating the interaction. Mutations in these critical residues disrupted the interaction between USP7 and EZH2. Furthermore, USP7 silencing and knockout experiments showed decreased EZH2 levels in HCT116 carcinoma cells. Finally, we demonstrated decreased H3K27Me3 levels in HCT116 USP7 knockout cells. These results indicate that USP7 interacts with EZH2 and regulates both its stability and function.

Synthesis of defined mono-de-N-acetylated ß-(1→6)-N-acetyl-d-glucosamine oligosaccharides to characterize PgaB hydrolase activity.

Many clinically-relevant biofilm-forming bacterial strains produce partially de-N-acetylated poly-ß-(1→6)-N-acetyl-d-glucosamine (dPNAG) as an exopolysaccharide. In Gram-negative bacteria, the periplasmic protein PgaB is responsible for partial de-N-acetylation of PNAG prior to its export to the extracellular space. In addition to de-N-acetylase activity found in the N-terminal domain, PgaB contains a C-terminal hydrolase domain that can disrupt dPNAG-dependent biofilms and hydrolyzes dPNAG but not fully acetylated PNAG. The role of this C-terminal domain in biofilm formation has yet to be determined in vivo. Further characterization of the enzyme's hydrolase activity has been hampered by a lack of specific dPNAG oligosaccharides. Here, we report the synthesis of a defined mono de-N-acetylated dPNAG penta- and hepta-saccharide. Using mass spectrometry analysis and a fluorescence-based thin-layer chromatography (TLC) assay, we found that our defined dPNAG oligosaccharides are hydrolase substrates. In addition to the expected cleavage site, two residues to the reducing side of the de-N-acetylated residue, minor cleavage products on the non-reducing side of the de-N-acetylation site were observed. These findings provide quantitative data to support how PNAG is processed in Gram-negative bacteria.

PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-ß(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.

Poly-ß(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides.

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the biosynthesis of NAD(+) and NaAD(+). The crystal structure of NMNAT from Methanobacterium thermoautotrophicum complexed with NAD(+) and SO4(2-) revealed the active-site residues involved in binding and catalysis. Site-directed mutagenesis was used to further characterize the roles played by several of these residues. Arg11 and Arg136 were implicated in binding the phosphate groups of the ATP substrate. Both of these residues were mutated to lysine individually. Arg47 does not interact with either NMN or ATP substrates directly, but was deemed to play a role in binding as it is proximal to Arg11 and Arg136. Arg47 was mutated to lysine and glutamic acid. Surprisingly, when expressed in Escherichia coli all of these NMNAT mutants trapped a molecule of NADP(+) in their active sites. This NADP(+) was bound in a conformation that was quite different from that displayed by NAD(+) in the native enzyme complex. When NADP(+) was co-crystallized with wild-type NMNAT, the same structural arrangement was observed. These studies revealed a different conformation of NADP(+) in the active site of NMNAT, indicating plasticity of the active site.

Characterization of Vis Toxin, a Novel ADP-Ribosyltransferase from Vibrio splendidus.

Vis toxin was identified by a bioinformatics strategy as a putative virulence factor produced by Vibrio splendidus with mono-ADP-ribosyltransferase activity. Vis was purified to homogeneity as a 28 kDa single-domain enzyme and was shown to possess NAD(+)-glycohydrolase [KM(NAD(+)) = 276 ± 12 µM] activity and with an R-S-E-X-E motif; it targets arginine-related compounds [KM(agmatine) = 272 ± 18 mM]. Mass spectrometry analysis revealed that Vis labels l-arginine with ADP-ribose from the NAD(+) substrate at the amino nitrogen of the guanidinium side chain. Vis is toxic to yeast when expressed in the cytoplasm under control of the CUP1 promotor, and catalytic variants lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. Several small molecule inhibitors were identified from a virtual screen, and the most potent compounds were found to inhibit the transferase activity of the enzyme with Ki values ranging from 25 to 134 µM. Inhibitor compound M6 bears the necessary attributes of a solid candidate as a lead compound for therapeutic development. Vis toxin was crystallized, and the structures of the apoenzyme (1.4 Å) and the enzyme bound with NAD(+) (1.8 Å) and with the M6 inhibitor (1.5 Å) were determined. The structures revealed that Vis represents a new subgroup within the mono-ADP-ribosyltransferase toxin family.

Crystal Structure of USP7 Ubiquitin-like Domains with an ICP0 Peptide Reveals a Novel Mechanism Used by Viral and Cellular Proteins to Target USP7.

Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.

Deubiquitinases and the new therapeutic opportunities offered to cancer.

Deubiquitinases (DUBs) play important roles and therefore are potential drug targets in various diseases including cancer and neurodegeneration. In this review, we recapitulate structure-function studies of the most studied DUBs including USP7, USP22, CYLD, UCHL1, BAP1, A20, as well as ataxin 3 and connect them to regulatory mechanisms and their growing protein interaction networks. We then describe DUBs that have been associated with endocrine carcinogenesis with a focus on prostate, ovarian, and thyroid cancer, pheochromocytoma, and adrenocortical carcinoma. The goal is enhancing our understanding of the connection between dysregulated DUBs and cancer to permit the design of therapeutics and to establish biomarkers that could be used in diagnosis and prognosis.

The structure and regulation of magnesium selective ion channels.

The magnesium ion (Mg(2+)) is the most abundant divalent cation within cells. In man, Mg(2+)-deficiency is associated with diseases affecting the heart, muscle, bone, immune, and nervous systems. Despite its impact on human health, little is known about the molecular mechanisms that regulate magnesium transport and storage. Complete structural information on eukaryotic Mg(2+)-transport proteins is currently lacking due to associated technical challenges. The prokaryotic MgtE and CorA magnesium transport systems have recently succumbed to structure determination by X-ray crystallography, providing first views of these ubiquitous and essential Mg(2+)-channels. MgtE and CorA are unique among known membrane protein structures, each revealing a novel protein fold containing distinct arrangements of ten transmembrane-spanning α-helices. Structural and functional analyses have established that Mg(2+)-selectivity in MgtE and CorA occurs through distinct mechanisms. Conserved acidic side-chains appear to form the selectivity filter in MgtE, whereas conserved asparagines coordinate hydrated Mg(2+)-ions within the selectivity filter of CorA. Common structural themes have also emerged whereby MgtE and CorA sense and respond to physiologically relevant, intracellular Mg(2+)-levels through dedicated regulatory domains. Within these domains, multiple primary and secondary Mg(2+)-binding sites serve to staple these ion channels into their respective closed conformations, implying that Mg(2+)-transport is well guarded and very tightly regulated. The MgtE and CorA proteins represent valuable structural templates to better understand the related eukaryotic SLC41 and Mrs2-Alr1 magnesium channels. Herein, we review the structure, function and regulation of MgtE and CorA and consider these unique proteins within the expanding universe of ion channel and transporter structural biology.

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation.

Magnesium ions (Mg(2+)) are essential for life, but the mechanisms regulating their transport into and out of cells remain poorly understood. The CorA-Mrs2-Alr1 superfamily of Mg(2+) channels represents the most prevalent group of proteins enabling Mg(2+) ions to cross membranes. Thermotoga maritima CorA (TmCorA) is the only member of this protein family whose complete 3D fold is known. Here, we report the crystal structure of a mutant in the presence and absence of divalent ions and compare it with previous divalent ion-bound TmCorA structures. With Mg(2+) present, this structure shows binding of a hydrated Mg(2+) ion to the periplasmic Gly-Met-Asn (GMN) motif, revealing clues of ion selectivity in this unique channel family. In the absence of Mg(2+), TmCorA displays an unexpected asymmetric conformation caused by radial and lateral tilts of protomers that leads to bending of the central, pore-lining helix. Molecular dynamics simulations support these movements, including a bell-like deflection. Mass spectrometric analysis confirms that major proteolytic cleavage occurs within a region that is selectively exposed by such a bell-like bending motion. Our results point to a sequential allosteric model of regulation, where intracellular Mg(2+) binding locks TmCorA in a symmetric, transport-incompetent conformation and loss of intracellular Mg(2+) causes an asymmetric, potentially influx-competent conformation of the channel.

Computer-aided structure analysis of an epimerized dehydroepiandrosterone derivative and its biological effect in a model of reactive gliosis.

The naturally occurring steroid dehydroepiandrosterone (DHEA) is reported to reduce glial fibrillary acidic protein (GFAP) overexpression in a model of reactive gliosis due to its conversion to estradiol by the enzyme aromatase. In the present study we examined the biological effect of a new epimerized derivative of DHEA, 16alpha-iodomethyl-13alpha-dehydroepiandrosterone derivative (16alpha-iodomethyl-13alpha-DHEAd, 16alpha-iodomethyl-13alpha-androst-5-en-3beta,17beta-diol), using the same model system, and compared the 3D structure of this molecule with that of DHEA and two steroidal type aromatase inhibitors, formestane and exemestane. The synthetic compound, in contrast to the reported effect of DHEA, was able to reduce GFAP overexpression only if the enzyme aromatase was inhibited. Data obtained from computational calculations fortified by X-ray crystallography revealed that contrary to the nearly planar sterane framework of DHEA, the synthetic derivative 16alpha-iodomethyl-13alpha-DHEAd has a bent sterane skeleton, resulting in a 3D structure that is similar to that of formestane or exemestane. Moreover, 16alpha-iodomethyl-13alpha-DHEAd resulted to be metabolically more stable than DHEA. The results suggest that epimerization of the sterane skeleton of DHEA inclines the plane of the D ring, leading to a significantly altered biological activity. The synthetic molecule has a DHEA-like effect on GFAP overexpression when the enzyme aromatase is inhibited and the naturally occurring DHEA is ineffective in this respect. On the other hand, based on their structural similarity it can be hypothesized that 16alpha-iodomethyl-13alpha-DHEAd applied alone might have a biological effect similar to that of formestane or exemestane.

Interaction of an echinomycin-DNA complex with manganese ions.

The crystal structure of an echinomycin-d(ACGTACGT) duplex interacting with manganese(II) was solved by Mn-SAD using in-house data and refined to 1.1 A resolution against synchrotron data. This complex crystallizes in a different space group compared with related complexes and shows a different mode of base pairing next to the bis-intercalation site, suggesting that the energy difference between Hoogsteen and Watson-Crick pairing is rather small. The binding of manganese to N7 of guanine is only possible because of DNA unwinding induced by the echinomycin, which might help to explain the mode of action of the drug.

T-muurolol sesquiterpenes from the marine Streptomyces sp. M491 and revision of the configuration of previously reported amorphanes.

Two new sesquiterpenes, 15-hydroxy-T-muurolol (3d) and 11,15-dihydroxy-T-muurolol (3e), along with the plant cadinenes T-muurolol (3f) and 3alpha-hydroxy-T-muurolol (3g), were isolated from the marine-derived Streptomyces sp. M491. Their absolute configuration was established via NMR spectroscopy and X-ray crystallography of 3-oxo-T-muurolol (3a), which was reisolated from this strain. In addition, the absolute configuration of further sesquiterpenes previously reported from this strain was revised. These products were tested for their cytotoxicity against 37 human tumor cell lines using the MTT method. Only 3d was cytotoxic against a range of human tumor cell lines with a mean IC50 of 6.7 microg/mL.

Crystal structure of trioxacarcin A covalently bound to DNA.

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule 'caught in the act' after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 A resolution by MAD phasing employing brominated oligonucleotides. The DNA-drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3'-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.

Enantio- and diastereoselective synthesis of duocarmycine-based prodrugs for a selective treatment of cancer by epoxide opening.

For the enantio- und diastereoselective synthesis of the prodrug 2, the N-tert-butyloxycarbonyl-protected amine 7 was alkylated with the enantiopure epoxide 14 to give the amide 10. A regio- and facial-selective metal-mediated cyclisation by using a cuprate led to 17 with an inversion of configuration at C10. Subsequent transformation of the hydroxy group in 17 by using the Appel procedure afforded (1S,10R)-9 with an unusual double inversion owing to neighbouring-group participation of the N-tert-butoxycarbonyl group. (1S,10R)-9 is the key intermediate in the synthesis of the prodrug 2, which has been developed for a selective treatment of cancer based on the antibody-directed enzyme prodrug therapy as an analogue of the natural antibiotic duocarmycine SA (1).

Rhenium oxo compounds containing eta2-pyrazolate ligands.

Reaction of potassium salts of sterically demanding pyrazolates (pz = bis-3,5-tert-butylpyrazolate, pz= bis-3,5-tert-butyl-4-methylpyrazolate) with Re2O7 affords soluble eta2-pyrazolate complexes of the type [(eta2-pz)ReO3(THF)n](1: pz, n= 1 and 2: pz, n= 0). They were characterized by spectroscopic methods and by X-ray crystallography confirming the eta2-coordinate ligands. Complex 1 employing the ligand with a proton in the 4-position retains one molecule of THF, whereas the additional methyl group in 2 leads to the base-free compound 2. Compound 1 reacts with pyridine and 3,5-dimethylpyridine to form Lewis base adducts of the type [(eta2-pz)ReO3(L)](3: L = py; 4: L = 3,5-Me2py). The pronounced sensitivity towards water of these complexes is demonstrated by the reaction of 1 with one equivalent of water forming the corresponding pyrazolium perrhenate [ReO4][pzH2](5). Its solid state structure shows a hydrogen bonded dimeric assembly. Catalytic activity of 1 is established in oxygen atom transfer-reactions (OAT) from dimethylsulfoxide to triphenylphosphine, and in epoxidations of cyclooctene employing bis(trimethylsilyl) peroxide (BTSP).