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  • 1
    Publication Date: 2013-03-22
    Description: Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an alpha-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host-pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Gatsogiannis, Christos -- Lang, Alexander E -- Meusch, Dominic -- Pfaumann, Vanda -- Hofnagel, Oliver -- Benz, Roland -- Aktories, Klaus -- Raunser, Stefan -- England -- Nature. 2013 Mar 28;495(7442):520-3. doi: 10.1038/nature11987. Epub 2013 Mar 20.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23515159" target="_blank"〉PubMed〈/a〉
    Keywords: ADP Ribose Transferases/chemistry/metabolism/ultrastructure ; Animals ; Bacterial Proteins/chemistry/*metabolism/ultrastructure ; Bacterial Toxins/chemistry/*metabolism ; Cell Membrane/metabolism ; Cryoelectron Microscopy ; Cytoplasm/metabolism ; Host-Pathogen Interactions ; Insects/cytology/metabolism/microbiology ; Models, Biological ; Models, Molecular ; Photorhabdus/*metabolism/pathogenicity/ultrastructure ; Pore Forming Cytotoxic Proteins/chemistry/*metabolism/ultrastructure ; Protein Conformation ; Protein Transport
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
    Publication Date: 2014-02-28
    Description: Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly understood. Here we report the first, to our knowledge, high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex. The structures reveal that, in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed beta-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation. Our results allow us to understand key steps of infections involving Tc toxins at the molecular level.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Meusch, Dominic -- Gatsogiannis, Christos -- Efremov, Rouslan G -- Lang, Alexander E -- Hofnagel, Oliver -- Vetter, Ingrid R -- Aktories, Klaus -- Raunser, Stefan -- England -- Nature. 2014 Apr 3;508(7494):61-5. doi: 10.1038/nature13015. Epub 2014 Feb 23.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany [2]. ; Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universitat Freiburg, 79104 Freiburg, Germany. ; Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany. ; Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany. ; 1] Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universitat Freiburg, 79104 Freiburg, Germany [2] BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universitat Freiburg, 79104 Freiburg, Germany. ; 1] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany [2] Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Thielallee 63, 14195 Berlin, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24572368" target="_blank"〉PubMed〈/a〉
    Keywords: ADP Ribose Transferases/metabolism ; Bacterial Toxins/*chemistry/*metabolism ; Binding Sites ; Cell Membrane/metabolism ; Crystallography, X-Ray ; Host Specificity ; Hydrogen-Ion Concentration ; Models, Molecular ; Neuraminidase/chemistry ; Photorhabdus/*chemistry ; Porosity ; Protein Structure, Tertiary ; Protein Subunits/chemistry/metabolism ; Protein Transport ; Protein Unfolding ; Structure-Activity Relationship
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 3
    Publication Date: 2012-06-02
    Description: Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677693/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677693/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hernandez, Javier M -- Stein, Alexander -- Behrmann, Elmar -- Riedel, Dietmar -- Cypionka, Anna -- Farsi, Zohreh -- Walla, Peter J -- Raunser, Stefan -- Jahn, Reinhard -- 3P01GM072694-05S1/GM/NIGMS NIH HHS/ -- P01 GM072694/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 2012 Jun 22;336(6088):1581-4. doi: 10.1126/science.1221976. Epub 2012 May 31.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/22653732" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Lipid Bilayers/chemistry/*metabolism ; *Liposomes/chemistry/metabolism ; *Membrane Fusion ; Protein Binding ; Protein Conformation ; Rats ; SNARE Proteins/chemistry/*metabolism ; Vesicle-Associated Membrane Protein 2/metabolism
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 2014-12-04
    Description: Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 A in complex with tropomyosin at a resolution of 6.5 A, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477711/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477711/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉von der Ecken, Julian -- Muller, Mirco -- Lehman, William -- Manstein, Dietmar J -- Penczek, Pawel A -- Raunser, Stefan -- R01 60635/PHS HHS/ -- R01 GM060635/GM/NIGMS NIH HHS/ -- R37HL036153/HL/NHLBI NIH HHS/ -- U54 094598/PHS HHS/ -- U54 GM094598/GM/NIGMS NIH HHS/ -- England -- Nature. 2015 Mar 5;519(7541):114-7. doi: 10.1038/nature14033. Epub 2014 Dec 1.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany. ; Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany. ; Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, USA. ; Department of Biochemistry and Molecular Biology, The University of Texas, Houston Medical School, Houston, Texas 77030, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25470062" target="_blank"〉PubMed〈/a〉
    Keywords: Actins/*chemistry/genetics/*metabolism ; Adenosine Diphosphate/metabolism ; Animals ; Cryoelectron Microscopy ; Magnesium/metabolism ; Mice ; Models, Molecular ; Mutation/genetics ; Protein Conformation ; Rabbits ; Static Electricity ; Tropomyosin/*chemistry/genetics/*metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 5
    Publication Date: 2014-12-04
    Description: Muscle contraction is initiated by the release of calcium (Ca(2+)) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 A. We show that the cytoplasmic moiety of RyR1 contains two large alpha-solenoid domains and several smaller domains, with folds suggestive of participation in protein-protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Efremov, Rouslan G -- Leitner, Alexander -- Aebersold, Ruedi -- Raunser, Stefan -- England -- Nature. 2015 Jan 1;517(7532):39-43. doi: 10.1038/nature13916. Epub 2014 Dec 1.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany [2] Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), 1050 Brussels, Belgium [3] Structural Biology Brussels, Vrije Universiteit Brussel (VUB), 1050 Brussels, Belgium. ; Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland. ; 1] Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland [2] Faculty of Science, University of Zurich, 8057 Zurich, Switzerland. ; Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25470059" target="_blank"〉PubMed〈/a〉
    Keywords: Allosteric Regulation/drug effects ; Animals ; Calcium/deficiency/metabolism/pharmacology ; Cryoelectron Microscopy ; Cytoplasm/metabolism ; Hydrogen-Ion Concentration ; Inositol 1,4,5-Trisphosphate Receptors/chemistry ; Ion Channel Gating/drug effects ; Models, Molecular ; Protein Binding ; Protein Structure, Tertiary/drug effects ; Rabbits ; Ryanodine Receptor Calcium Release Channel/chemistry/*metabolism/*ultrastructure ; Tacrolimus Binding Protein 1A/chemistry/metabolism/ultrastructure
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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