The structural basis of flagellin detection by NAIP5 Tenthorey, Jeannette L.; Haloupek, Nicole; López-Blanco, José Ramón ...
Science (American Association for the Advancement of Science),
11/2017, Letnik:
358, Številka:
6365
Journal Article
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Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in ...plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form amultiprotein complex called an inflammasome. Here we report the cryo–electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.
Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related ...post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here, we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 10
µm
. These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue,
brain, and
to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.
Modularity of a carbon-fixing protein organelle Bonacci, Walter; Teng, Poh K; Afonso, Bruno ...
Proceedings of the National Academy of Sciences - PNAS,
01/2012, Letnik:
109, Številka:
2
Journal Article
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Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is ...known about their assembly and function in vivo. We show here that carboxysomes, CO2-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO2. Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO2 in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.
Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in ...vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca 2+ triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca 2+ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca 2+ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca 2+ sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca 2+ triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.
Abstract Stramenopile algae contribute significantly to global primary productivity, and one class, Eustigmatophyceae, is increasingly studied for applications in high-value lipid production. Yet ...much about their basic biology remains unknown, including the nature of an enigmatic, pigmented globule found in vegetative cells. Here, we present an in-depth examination of this “red body,” focusing on Nannochloropsis oceanica . During the cell cycle, the red body forms adjacent to the plastid, but unexpectedly it is secreted and released with the autosporangial wall following cell division. Shed red bodies contain antioxidant ketocarotenoids, and overexpression of a beta-carotene ketolase results in enlarged red bodies. Infrared spectroscopy indicates long-chain, aliphatic lipids in shed red bodies and cell walls, and UHPLC-HRMS detects a C32 alkyl diol, a potential precursor of algaenan, a recalcitrant cell wall polymer. We propose that the red body transports algaenan precursors from plastid to apoplast to be incorporated into daughter cell walls.
Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear heterooligomeric complexes, which in turn are able to polymerize into apolar filaments and higher-order ...structures. In budding yeast (
Saccharomyces cerevisiae) and other eukaryotes, proper septin organization is essential for processes that involve membrane remodeling, such as the execution of cytokinesis. In yeast, four septin subunits form a Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 heterooctameric rod that polymerizes into filaments thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin–membrane interactions, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microscopy. Using this methodology, we have acquired new insights into the potential effect of septin–membrane interactions on filament assembly and, more specifically, on the role of phosphoinositides. Our studies demonstrate that budding yeast septins interact specifically with phosphatidylinositol-4,5-
bisphosphate (PIP2) and indicate that the N terminus of Cdc10 makes a major contribution to the interaction of septin filaments with PIP2. Furthermore, we found that the presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions that prevent filament formation in solution or for mutants that prevent filament formation in solution. In the extreme case of septin complexes lacking the normally terminal subunit Cdc11 or the normally central Cdc10 doublet, the combination of the PIP2-containing monolayer and nucleotide permitted filament formation
in vitro via atypical Cdc12–Cdc12 and Cdc3–Cdc3 interactions, respectively.
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► PIP2-containing lipid monolayers promote a novel organization of septin filaments not observed in solution. ► PIP2-containing lipid monolayers dramatically enhance the formation of filaments, even for septin complexes lacking one of the septin proteins. ► The N terminus of Cdc10 is important for interaction with PIP2. ► Our
in vitro studies strongly suggest that the PIP2 present at the bud neck is important for septin organization
in situ.
Clicking into place: Cu‐free click chemistry is combined with the aldehyde tag protein modification strategy to produce heterobifunctional protein fusions. This method relies on linkers that utilize ...orthogonal triazole and oxime chemistries (see scheme) to expand on conjugation with the fGly residue and enables site‐specific protein conjugation to full‐length human antibodies.
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and ...the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.
A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step ...of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIID’s lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.
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► TFIID has distinct conformations with different positions of the TBP-containing lobe ► A rearranged state of TFIID interacts with promoter DNA in a TFIIA-dependent manner ► TFIID induces multiple topological changes on promoter DNA around the start site ► The different conformations of TFIID may serve as targets for regulatory factors
TFIID undergoes a radical structural rearrangement when it binds promoter DNA. The bound form identified in this study defines TFIID’s interaction with the upstream and downstream core promoter regions during a key step in transcription initiation.
Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their ...arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel "railroad tracks." Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures.
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