Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.

Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .

Structural basis for topological regulation of Tn3 resolvase.

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Site-specific DNA recombinases alter the connectivity of DNA by recognizing specific DNA sequences, then cutting the DNA strands and pasting them together in a new configuration. Such enzymes play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful biotechnology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. However, others have evolved elaborate mechanisms to ensure that only one of multiple possible recombination products is produced. Tn3 resolvase has long been known to be regulated by DNA topology­that is, it will cut and reconnect two target sequences only if they lie on the same DNA molecule, and if they are in the proper relative orientation. This study presents new structural and biochemical data that lead to a new, detailed model of the large protein­DNA complex formed by Tn3 resolvase and its cognate sites. This 3D model illustrates how DNA topology can be harnessed to regulate the activity of a recombinase and provides a basis for engineering Tn3 resolvase and related recombination systems as genome editing tools.

Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes.

The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.

Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy.

Interaction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D'D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D'D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII-VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D'D3 is covalently linked to an Fc domain of a B domain-deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D'D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII-VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF-D'/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.

Crystal structure of the Varkud satellite ribozyme.

The Varkud satellite (VS) ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.

PanG, a new ketopantoate reductase involved in pantothenate synthesis.

Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation.1 Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases,2 while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55.3 While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited.4 For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent)6,7 and FAM122A5 (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.

Francisella tularensis RipA protein topology and identification of functional domains.

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1ß (IL-1ß) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function.

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis.

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1ß, IL-18, and TNF-α by resting macrophages. IL-1ß and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1ß and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.

A bivalent Epstein-Barr virus vaccine induces neutralizing antibodies that block infection and confer immunity in humanized mice.

Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers and, more recently, multiple sclerosis. Despite its prevalence and health impact, there are currently no vaccines or treatments. Four viral glycoproteins (gp), gp350 and gH/gL/gp42, mediate entry into the major sites of viral replication, B cells, and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and showed that it elicits potent neutralizing antibodies that protect against infection in vivo. We designed single-chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticle. Structural analysis revealed that single-chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild-type proteins, and the protein spikes were observed by electron microscopy. Single-chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and nonhuman primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123, or gH/gL+gp350D123 nanoparticles. Although all control animals were infected, only one mouse in each vaccine group that received immune IgG had detectable transient viremia. Furthermore, no EBV lymphomas were detected in immune animals. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans.

Effects of the putative transcriptional regulator IclR on Francisella tularensis pathogenesis.

Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intracellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis, respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 ΔiclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSΔiclR were equally cytotoxic and induced equivalent levels of interleukin-1ß expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and ΔiclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.

An ER translocon for multi-pass membrane protein biogenesis.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Cell membranes are structures that separate the interior of the cell from its environment and determine the cell's shape and the structure of its internal compartments. Nearly 25% of human genes encode transmembrane proteins that span the entire membrane from one side to the other, helping the membrane perform its roles. Transmembrane proteins are synthesized by ribosomes ­ protein-making machines ­ that are on the surface of a cell compartment called the endoplasmic reticulum. As the new protein is made by the ribosome, it enters the endoplasmic reticulum membrane where it folds into the correct shape. This process is best understood for proteins that span the membrane once. Despite decades of work, however, much less is known about how multi-pass proteins that span the membrane multiple times are made. A study from 2017 showed that a protein called TMCO1 is related to a group of proteins involved in making membrane proteins. TMCO1 has been linked to glaucoma, and mutations in it cause cerebrofaciothoracic dysplasia, a human disease characterized by severe intellectual disability, distinctive facial features, and bone abnormalities. McGilvray, Anghel et al. ­ including several of the researchers involved in the 2017 study ­ wanted to determine what TMCO1 does in the cell and begin to understand its role in human disease. McGilvray, Anghel et al. discovered that TMCO1, together with other proteins, is part of a new 'translocon' ­ a group of proteins that transports proteins into the endoplasmic reticulum membrane. Using a combination of biochemical, genetic and structural techniques, McGilvray, Anghel et al. showed that the translocon interacts with ribosomes that are synthesizing multi-pass proteins. The experiments revealed that the translocon is required for the production of a multi-pass protein called EAAT1, and it provides multiple ways for proteins to be inserted into and folded within the membrane. The findings of McGilvray, Anghel et al. reveal a previously unknown cellular machinery which may be involved in the production of hundreds of human multi-pass proteins. This work provides a framework for understanding how these proteins are correctly made in the membrane. Additionally, it suggests that human diseases caused by mutations in TMCO1 result from a defect in the production of multi-pass membrane proteins.

Environmental and intracellular regulation of Francisella tularensis ripA.

BACKGROUND: Francisella tularensis is a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia. RipA is a cytoplasmic membrane protein that is conserved among Francisella species and is required for intracellular growth. F. tularensis ripA deletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm. RESULTS: Further analysis of ripA with respect to environmental effects on the growth of mutant strains and expression levels revealed that RipA is required for optimal growth at pH 7.5 but not pH 6.5. Using a combination of RT-PCR, ripA-lacZ transcriptional and translational fusions, and a RipA-tetracysteine tag fusion protein we found that both ripA transcription and RipA protein levels were elevated in organisms grown at pH 7.5 as compared to organisms grown at pH 5.5. A number of genes, including iglA, that are required for intracellular growth are regulated by the transcriptional regulators MglA and SspA, and are induced upon infection of host cells. We quantified ripA and iglA expression at different stages of intracellular growth and found that the expression of each increased between 1 and 6 hours post infection. Given the similar intracellular expression patterns of ripA and iglA and that MglA and SspA are positive regulators of iglA we tested the impact of mglA and sspA deletions on ripA and iglA expression. In the deletion mutant strains iglA expression was reduced dramatically as expected, however ripA expression was increased over 2-fold. CONCLUSION: Expression of ripA is required for growth at neutral pH, is pH sensitive, and is responsive to the intracellular environment. The intracellular expression pattern of ripA coincided with iglA, which is positively regulated by MglA and SspA. However, in contrast to their positive impact on iglA expression, MglA and SspA negatively impacted ripA expression in vitro.

A conserved RNA structural motif for organizing topology within picornaviral internal ribosome entry sites.

Picornaviral IRES elements are essential for initiating the cap-independent viral translation. However, three-dimensional structures of these elements remain elusive. Here, we report a 2.84-Å resolution crystal structure of hepatitis A virus IRES domain V (dV) in complex with a synthetic antibody fragment-a crystallization chaperone. The RNA adopts a three-way junction structure, topologically organized by an adenine-rich stem-loop motif. Despite no obvious sequence homology, the dV architecture shows a striking similarity to a circularly permuted form of encephalomyocarditis virus J-K domain, suggesting a conserved strategy for organizing the domain architecture. Recurrence of the motif led us to use homology modeling tools to compute a 3-dimensional structure of the corresponding domain of foot-and-mouth disease virus, revealing an analogous domain organizing motif. The topological conservation observed among these IRESs and other viral domains implicates a structured three-way junction as an architectural scaffold to pre-organize helical domains for recruiting the translation initiation machinery.

RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival.

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.

Francisella tularensis invasion of lung epithelial cells.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, causes disseminating infections in humans and other mammalian hosts. Macrophages and other monocytes have long been considered the primary site of F. tularensis replication in infected animals. However, recently it was reported that F. tularensis also invades and replicates within alveolar epithelial cells following inhalation in a mouse model of tularemia. TC-1 cells, a mouse lung epithelial cell line, were used to study the process of F. tularensis invasion and intracellular trafficking within nonphagocytic cells. Live and paraformaldehyde-fixed F. tularensis live vaccine strain organisms associated with, and were internalized by, TC-1 cells at similar frequencies and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreased F. tularensis invasion, as did inhibitors of phosphatidylinositol 3-kinase and tyrosine kinase activity. Collectively, these results suggest that F. tularensis epithelial cell invasion is mediated by a preformed ligand on the bacterial surface and driven entirely by host cell processes. Once internalized, F. tularensis-containing endosomes associated with early endosome antigen 1 (EEA1) followed by lysosome-associated membrane protein 1 (LAMP-1), with peak coassociation frequencies occurring at 30 and 120 min postinoculation, respectively. By 2 h postinoculation, 70.0% (+/- 5.5%) of intracellular bacteria were accessible to antibody delivered to the cytoplasm, indicating vacuolar breakdown and escape into the cytoplasm.

Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif.

The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO3 bound forms of DIR2s at 2.0 Å and 1.8 Å resolution, respectively. DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO3 fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO3, and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.

Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal.

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.

Characterization of glycans on major histocompatibility complex class II molecules in channel catfish, Ictalurus punctatus.

The glycans associated with mammalian major histocompatibility complex (MHC) class II molecules have been studied extensively. Co-translational and post-translational addition of sugar molecules to proteins confers many structural and modulatory functions. In the present study we characterized the glycans associated with MHC class II molecules in the channel catfish to compare glycosylation patterns in a teleost to those known to occur in mammals. This study made use of enzymatic methods and two-dimensional (2D) gel electrophoresis to characterize the N-linked sugars. Unlike mammalian T cells which expressed complex N-linked sugars, channel catfish derived 28S T cells were found to express high-mannose/hybrid N-glycans on class II molecules. However studies with Endoglycosidase H in conjunction with cell surface labeling on peripheral blood leukocytes revealed that catfish possess the machinery to modify the intermediate high-mannose sugars to complex type sugars. Nonetheless, the majority of the class II cell surface glycoproteins were of the high-mannose type. Resolution of catfish MHC class II molecules by 2D gel analyses revealed multiple bands for class II beta chains whereas class II alpha chains focused as a single spot. Glycosylation in the channel catfish, a premier model system for studying the immune system of teleosts, has significant differences from the glycosylation patterns characterized in mammalian systems, likely with functional implications.