The NS1 protein of influenza B virus binds 5'-triphosphorylated dsRNA to suppress RIG-I activation and the host antiviral response.

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non-structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5'triphosphorylated double-stranded RNA (5'-ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response. Like RIG-I, NS1B-CTD preferentially binds blunt-end 5'ppp-dsRNA. NS1B-CTD also competes with RIG-I for binding 5'ppp-dsRNA, and thus suppresses activation of RIG-I's ATPase activity. Although the NS1B N-terminal domain also binds dsRNA, it utilizes a different binding mode and lacks 5'ppp-dsRNA end preferences. In cells infected with wild-type influenza B virus, RIG-I activation is inhibited. In contrast, RIG-I activation and the resulting phosphorylation of transcription factor IRF-3 are not inhibited in cells infected with a mutant virus encoding NS1B with a R208A substitution it its CTD that eliminates its 5'ppp-dsRNA binding activity. These results reveal a novel mechanism in which NS1B binds 5'ppp-dsRNA to inhibit the RIG-I antiviral response during influenza B virus infection, and open the door to new avenues for antiviral drug discovery.

Oligomeric interactions maintain active-site structure in a noncooperative enzyme family.

The evolutionary benefit accounting for widespread conservation of oligomeric structures in proteins lacking evidence of intersubunit cooperativity remains unclear. Here, crystal and cryo-EM structures, and enzymological data, demonstrate that a conserved tetramer interface maintains the active-site structure in one such class of proteins, the short-chain dehydrogenase/reductase (SDR) superfamily. Phylogenetic comparisons support a significantly longer polypeptide being required to maintain an equivalent active-site structure in the context of a single subunit. Oligomerization therefore enhances evolutionary fitness by reducing the metabolic cost of enzyme biosynthesis. The large surface area of the structure-stabilizing oligomeric interface yields a synergistic gain in fitness by increasing tolerance to activity-enhancing yet destabilizing mutations. We demonstrate that two paralogous SDR superfamily enzymes with different specificities can form mixed heterotetramers that combine their individual enzymological properties. This suggests that oligomerization can also diversify the functions generated by a given metabolic investment, enhancing the fitness advantage provided by this architectural strategy.

ZapG (YhcB/DUF1043), a novel cell division protein in gamma-proteobacteria linking the Z-ring to septal peptidoglycan synthesis.

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

Secure application of graphene in medicine.

OBJECTIVE: The objective of our study was to explore the possible graphene impact on microorganism growth as well as on laboratory animal overall condition. Materials and technique: The experiments applied samples of graphene three concentrations and two 15 × 15 mm quartz glasses one of which carrying deposited graphene lattice. We have also used 5% blood agar, thioglycollate broth, bacterial suspensions of standard turbidity containing pure clinical isolate of microorganisms. Three white male 6-month-old laboratory rats were used to estimate the graphene impact on the overall animal condition. RESULTS: Graphene did not contain any microorganisms, does not destroy erythrocytes placed within the artificial nutritional medium, graphene lattice did not add any properties to the quartz glasses which could allow the Proteus spread all over its surface. It was also established that graphene did not show any native antibacterial impact. No significant reaction was noticed in animals after graphene administration to laboratory rats neither at the injection spot nor at the overall level. CONCLUSION: Our data confirm the applicability of graphene both in scientific and practical biomedical purposes.

Cyclic oligomer design with de novo αß-proteins.

We have previously shown that monomeric globular αß-proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo-oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo-oligomer. The latter flexible backbone protocol generated designs with shape complementarity approaching that of native homo-oligomers, but experimental characterization showed that the fixed backbone designs were more stable and less aggregation prone. Designed C2 oligomers with ß-strand backbone interactions were structurally confirmed through x-ray crystallography and small-angle X-ray scattering (SAXS). In contrast, C3-C5 designed homo-oligomers with primarily nonpolar residues at interfaces all formed a range of oligomeric states. Taken together, our results suggest that for homo-oligomers formed from globular building blocks, improved structural specificity will be better achieved using monomers with increased shape complementarity and with more polar interfaces.

A community resource of experimental data for NMR / X-ray crystal structure pairs.

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.

The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS.

RAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and (19)F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase.

A general computational approach for repeat protein design.

Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1Å root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds.

Structure of the DNA-binding and RNA-polymerase-binding region of transcription antitermination factor λQ.

The bacteriophage λ Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, λQ binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. λQ binds to the paused early-elongation complex through interactions between λQ and two regions of RNA polymerase: region 4 of the σ(70) subunit and the flap region of the ß subunit. We present the 2.1 Å resolution crystal structure of a portion of λQ containing determinants for interaction with DNA, interaction with region 4 of σ(70), and interaction with the ß flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which λQ alters the functional properties of the transcription elongation complex.

Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of ß-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.

Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

Crystal structure of a catalytically active GG(D/E)EF diguanylate cyclase domain from Marinobacter aquaeolei with bound c-di-GMP product.

Recent studies of signal transduction in bacteria have revealed a unique second messenger, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which regulates transitions between motile states and sessile states, such as biofilms. C-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC). The catalytic activity of DGCs depends on a conserved GG(D/E)EF domain, usually part of a larger multi-domain protein organization. The domains other than the GG(D/E)EF domain often control DGC activation. This paper presents the 1.83 Å crystal structure of an isolated catalytically competent GG(D/E)EF domain from the A1U3W3_MARAV protein from Marinobacter aquaeolei. Co-crystallization with GTP resulted in enzymatic synthesis of c-di-GMP. Comparison with previously solved DGC structures shows a similar orientation of c-di-GMP bound to an allosteric regulatory site mediating feedback inhibition of the enzyme. Biosynthesis of c-di-GMP in the crystallization reaction establishes that the enzymatic activity of this DGC domain does not require interaction with regulatory domains.

Human retinoblastoma binding protein 9, a serine hydrolase implicated in pancreatic cancers.

Human retinoblastoma binding protein 9 (RBBP9) is an interacting partner of the retinoblastoma susceptibility protein (Rb). RBBP9 is a tumor-associated protein required for pancreatic neoplasia, affects cell cycle control, and is involved in the TGF-ß signalling pathway. Sequence analysis suggests that RBBP9 belongs to the α/ß hydrolase superfamily of enzymes. The serine hydrolase activity of RBBP9 is required for development of pancreatic carcinomas in part by inhibiting TGF-ß antiproliferative signaling through suppressing Smad2/3 phosphorylation. The crystal structure of human RBBP9 confirms the α/ß hydrolase fold, with a six-stranded parallel ß-sheet flanked by α helixes. The structure of RBBP9 resembles that of the YdeN protein from Bacillus subtilis, which is suggested to have carboxylesterase activity. RBBP9 contains a Ser75-His165-Asp138 catalytic triad, situated in a prominent pocket on the surface of the protein. The side chains of the LxCxE sequence motif that is important for interaction with Rb is mostly buried in the structure. Structure- function studies of RBBP9 suggest possible routes for novel cancer drug discovery programs.

UNC119 is required for G protein trafficking in sensory neurons.

UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like ß-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.

Improved molecular replacement by density- and energy-guided protein structure optimization.

Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

Solution NMR and X-ray crystal structures of membrane-associated Lipoprotein-17 domain reveal a novel fold.

The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0_UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a "domain of unknown function" (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0_UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 Å. The two structures are in good agreement with each other. The domain structure features three α-helices, α1 through α3, and five ß-strands. Strands ß1/ß2, ß3/ß4, ß4/ß5 are anti-parallel to each other. Strands ß1and ß2 are orthogonal to strands ß3, ß4, ß5, while helix α3 is formed between the strands ß3 and ß4. One-turn helix α2 is formed between the strands ß1 and ß2, while helix α1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0_UREPA, indicating that it is a novel protein fold.

Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.

A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma-vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.8 kHz provides capability for collecting multiple spectra from each particle-induced transient ion cloud, typically of 200-300 micros duration. It is shown that the transients can be resolved and characterized individually at a peak frequency of 1100 particles per second. Design considerations and optimization data are presented. The figures of merit of the instrument are measured under standard inductively coupled plasma (ICP) operating conditions (<3% cerium oxide ratio). At mass resolution (full width at half-maximum) M/DeltaM > 900 for m/z = 159, the sensitivity with a standard sample introduction system of >1.4 x 10(8) ion counts per second per mg L(-1) of Tb and an abundance sensitivity of (6 x 10(-4))-(1.4 x 10(-3)) (trailing and leading masses, respectively) are shown. The mass range (m/z = 125-215) and abundance sensitivity are sufficient for elemental immunoassay with up to 60 distinct available elemental tags. When <15 elemental tags are used, a higher sensitivity mode at lower resolution (M/DeltaM > 500) can be used, which provides >2.4 x 10(8) cps per mg L(-1) of Tb, at (1.5 x 10(-3))-(5.0 x 10(-3)) abundance sensitivity. The real-time simultaneous detection of multiple isotopes from individual 1.8 microm polystyrene beads labeled with lanthanides is shown. A real time single cell 20 antigen expression assay of model cell lines and leukemia patient samples immuno-labeled with lanthanide-tagged antibodies is presented.

Crystal structure of AGR_C_4470p from Agrobacterium tumefaciens.

We report here the crystal structure at 2.0 A resolution of the AGR_C_4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR_C_4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR_C_4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR_C_4470p in E. coli, in addition to the ChuS protein.

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-A resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.