3D structure of three jumbo phage heads Neumann, Emmanuelle; Kawasaki, Takeru; Effantin, Grégory ...
Journal of general virology,
11/2020, Letnik:
101, Številka:
11
Journal Article
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Jumbo phages are bacteriophages that carry more than 200 kbp of DNA. In this study we characterized two jumbo phages (ΦRSL2 and ΦXacN1) and one semi-jumbo phage (ΦRP13) at the structural level by ...cryo-electron microscopy. Focusing on their capsids, three-dimensional structures of the heads at resolutions ranging from 16 to 9 Å were calculated. Based on these structures we determined the geometrical basis on which the icosahedral capsids of these phages are constructed, which includes the accessory and decorative proteins that complement them. A triangulation number novel to
(ΦRP13;
=21) was discovered as well as two others, which are more common for jumbo phages (
=27 and
=28). Based on one of the structures we also provide evidence that accessory or decorative proteins are not a prerequisite for maintaining the structural integrity of very large capsids.
Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical ...properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields Phi (respectively, 0.38 and 0.25) and longer fluorescence lifetimes tau (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (Phi=0.28, tau=1.0 ns) and Cy7 (Phi=0.13, tau=0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging.
TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding ...sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules.
The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, ...respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that "zipper up" antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter.
The acentrosomal plant mitotic spindle is uniquely structured in that it lacks opposing centrosomes at its poles and is equipped with a connective preprophase band that regulates the spatial ...framework for spindle orientation and mobility. These features are supported by specialized microtubule-associated proteins and motors. Here, we show that Arabidopsis thaliana MAP65-4, a non-motor microtubule associated protein (MAP) that belongs to the evolutionarily conserved MAP65 family, specifically associates with the forming mitotic spindle during prophase and with the kinetochore fibers from prometaphase to the end of anaphase. In vitro, MAP65-4 induces microtubule (MT) bundling through the formation of cross-bridges between adjacent MTs both in polar and antipolar orientations. The association of MAP65-4 with an MT bundle is concomitant with its elongation. Furthermore, MAP65-4 modulates the MT dynamic instability parameters of individual MTs within a bundle, mainly by decreasing the frequency of catastrophes and increasing the frequency of rescue events, and thereby supports the progressive lengthening of MT bundles over time. These properties are in line with its role of initiating kinetochore fibers during prospindle formation.
Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of Fe-S clusters) ...often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 Fe
S
-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.
The α and β subunits of the human mitochondrial trifunctional protein (TFP), the multienzyme complex involved in fatty acid β-oxidation, were coexpressed in Escherichia coli and purified to ...homogeneity by nickel affinity chromatography. The resulting α/His-β construct was analyzed by gel filtration, sedimentation velocity, and electron microscopy, indicating a predominance of α2β2 and α4β4 complexes, with higher order oligomers. Electron microscopy indicated that the elementary species α2β2 had overall structural similarity with its bacterial homologue. As shown by cosedimentation and surface plasmon resonance analyses, recombinant TFP interacted strongly with cardiolipin and phosphatidylcholine, suggesting that the natural complex associates with the inner mitochondrial membrane through direct interactions with phospholipids. Recombinant TFP displayed 2-enoyl-CoA hydratase (ECH), l-3-hydroxyacyl-CoA dehydrogenase (HACD), and 3-ketoacyl-CoA thiolase (KACT) activities, and ECH and HACD each reached equilibrium when the downstream enzymes (HACD and KACT, respectively) were made inactive, indicating feed-back inhibition. The KACT activity was optimal at pH 9.5, sensitive to ionic strength, and inhibited at concentrations of its substrate 3-ketohexadecanoyl-CoA >5 μM. Its kinetic constants (k cat = 169 s−1, K m = 4 μM) were consistent with those determined previously on a purified porcine TFP preparation. Using different assays, trimetazidine, an efficient antiaginal agent, had no significant inhibitory effect on any of the three enzymatic activities of the recombinant TFP preparation, in contrast with other reports. This study provides the first detailed structural and functional characterization of a recombinant human TFP preparation and opens the way to in-depth analyses through site-directed mutagenesis.
Type III secretion (T3S) systems play key roles in pathogenicity of many Gram-negative bacteria and are employed to inject toxins directly into the cytoplasm of target cells. They are composed of ...over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which toxins travel. The PscF protein is the main component of the Pseudomonas aeruginosa T3S needle. Here we demonstrate that PscF, when purified on its own, is able to form needle-like fibers of 8 nm in width and >1 μm in length. In addition, we demonstrate for the first time that the T3S needle subunit requires two cytoplasmic partners, PscE and PscG, in P. aeruginosa, which trap PscF in a ternary, 1:1:1 complex, thus blocking it in a monomeric state. Knock-out mutants deficient in PscE and PscG are non-cytotoxic, lack PscF, and are unable to export PscF encoded extrachromosomally. Temperature-scanning circular dichroism measurements show that the PscE-PscF-PscG complex is thermally stable and displays a cooperative unfolding/refolding pattern. Thus, PscE and PscG prevent PscF from polymerizing prematurely in the P. aeruginosa cytoplasm and keep it in a secretion prone conformation, strategies which may be shared by other pathogens that employ the T3S system for infection.
Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid
protein VP2 is expressed in the baculovirus-insect cell system it ...assembles as core-like particles. The amino terminus region
of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP.
We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the
green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2
auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence
of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron
microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible
to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and
then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow
in real time the entry process of rotavirus and that chimeric VLP could be envisaged as ânanoboxesâ carrying macromolecules
to living cells.
Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The ...structure of the empty capsid has been determined to a resolution of better than 15 Å by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 Å diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA.
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