scholarly journals The pore conformation of lymphocyte perforin

2021 ◽  
Author(s):  
Marina Ivanova ◽  
Natalya Lukoyanova ◽  
Sony Malhotra ◽  
Maya Topf ◽  
Joseph A Trapani ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Target Cell ◽  
Pore Formation ◽  
Electron Tomography ◽  
Membrane Surface ◽  
Membrane Insertion ◽  
Cytotoxic Lymphocytes ◽  
Cryo Electron Tomography ◽  
Cancerous Cells ◽  
Β Sheet

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with pro-apoptotic granzymes, to bind to the plasma membrane of the target cell where it oligomerises and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here we present a 4 Å resolution cryo-EM structure of the perforin pore, revealing new inter- and intra-molecular interactions stabilising the pore. During assembly and pore formation, the helix-turn-helix motif at the bend in the central β-sheet moves away from the bend to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerisation and membrane insertion, and explain how several pathogenic mutations affect function.

scholarly journals Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-of-function toxicity

2021 ◽  
Vol 5 (3) ◽  
pp. e202101185
Author(s):  
Irene Riera-Tur ◽  
Tillman Schäfer ◽  
Daniel Hornburg ◽  
Archana Mishra ◽  
Miguel da Silva Padilha ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Protein Trafficking ◽  
Cell Biology ◽  
Electron Tomography ◽  
Lysosomal Storage ◽  
Gain Of Function ◽  
Cryo Electron Tomography ◽  
A Subunit ◽  
Late Stages ◽  
Β Sheet

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like β-sheet proteins (β proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, β proteins interact with and sequester AP-3 μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.

scholarly journals Hijacking the Fusion Complex of Human Parainfluenza Virus as an Antiviral Strategy

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
T. C. Marcink ◽  
E. Yariv ◽  
K. Rybkina ◽  
V. Más ◽  
F. T. Bovier ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Target Cell ◽  
Electron Tomography ◽  
Cell Receptor ◽  
Parainfluenza Virus ◽  
F Protein ◽  
Highly Active ◽  
Viral Particles ◽  
Virtual Modeling ◽  
Human Parainfluenza Virus

ABSTRACT The receptor binding protein of parainfluenza virus, hemagglutinin-neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein prior to receptor engagement of HN at the host cell membrane inactivate the virus. We previously identified small molecules that interact with HN and induce it to activate F in an untimely fashion, validating a new antiviral strategy. To obtain highly active pretriggering candidate molecules we carried out a virtual modeling screen for molecules that interact with sialic acid binding site II on HN, which we propose to be the site responsible for activating F. To directly assess the mechanism of action of one such highly effective new premature activating compound, PAC-3066, we use cryo-electron tomography on authentic intact viral particles for the first time to examine the effects of PAC-3066 treatment on the conformation of the viral F protein. We present the first direct observation of the conformational rearrangement induced in the viral F protein. IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), upon binding to its cell receptor, triggers conformational changes in the fusion protein (F). This action of HN activates F to reach its fusion-competent state. Using small molecules that interact with HN, we can induce the premature activation of F and inactivate the virus. To obtain highly active pretriggering compounds, we carried out a virtual modeling screen for molecules that interact with a sialic acid binding site on HN that we propose to be the site involved in activating F. We use cryo-electron tomography of authentic intact viral particles for the first time to directly assess the mechanism of action of this treatment on the conformation of the viral F protein and present the first direct observation of the induced conformational rearrangement in the viral F protein.

scholarly journals Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry

2016 ◽  
Vol 90 (17) ◽  
pp. 7618-7627 ◽  
Author(s):  
Erin E. H. Tran ◽  
Elizabeth A. Nelson ◽  
Pranay Bonagiri ◽  
James A. Simmons ◽  
Charles J. Shoemaker ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Structural Information ◽  
Electron Tomography ◽  
West African ◽  
Functional Studies ◽  
Cryo Electron Tomography ◽  
Niemann Pick ◽  
Viruslike Particles

ABSTRACTZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1+endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env.IMPORTANCEThe recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed—embedded in the membrane and present at high density—on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

scholarly journals In SituConformational Changes of theEscherichia coliSerine Chemoreceptor in Different Signaling States

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Wen Yang ◽  
C. Keith Cassidy ◽  
Peter Ames ◽  
Christoph A. Diebolder ◽  
Klaus Schulten ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Electron Tomography ◽  
Dynamics Simulation ◽  
Structural Constraints ◽  
Content Type ◽  
Cryo Electron Tomography ◽  
Structural Differences ◽  
Coupling Protein

ABSTRACTTsr, the serine chemoreceptor inEscherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associatein vivowith the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCEInEscherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of theE. coliserine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

scholarly journals The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling During Infection

Science ◽  
2013 ◽  
Vol 339 (6119) ◽  
pp. 576-579 ◽  
Author(s):  
Bo Hu ◽  
William Margolin ◽  
Ian J. Molineux ◽  
Jun Liu
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Cell Surface Receptor ◽  
Dna Ejection ◽  
Phage Tail ◽  
Cryo Electron Tomography ◽  
Surface Receptor ◽  
Host Cell Surface ◽  
Tail Fibers

Adsorption and genome ejection are fundamental to the bacteriophage life cycle, yet their molecular mechanisms are not well understood. We used cryo–electron tomography to capture T7 virions at successive stages of infection ofEscherichia coliminicells at ~4-nm resolution. The six phage tail fibers were folded against the capsid, extending and orienting symmetrically only after productive adsorption to the host cell surface. Receptor binding by the tail triggered conformational changes resulting in the insertion of an extended tail, which functions as the DNA ejection conduit into the cell cytoplasm. After ejection, the extended phage tail collapsed or disassembled, which allowed resealing of the infected cell membrane. These structural studies provide a detailed series of intermediates during phage infection.

scholarly journals In situ structure and priming mechanism of the rhoptry secretion system in Plasmodium revealed by cryo-electron tomography

2022 ◽  
Author(s):  
Matthew Martinez ◽  
William David Chen ◽  
Marta Cova ◽  
Petra Andrea Molnár ◽  
Shrawan Kumar Mageswaran ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Secretion System ◽  
Host Cells ◽  
Apicomplexan Parasites ◽  
Significant Difference ◽  
Cryo Electron Tomography ◽  
Parasite Plasmodium Falciparum ◽  
Priming Process

Apicomplexan parasites secrete the contents of rhoptries into host cells to permit their invasion and establishment of an infectious niche. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is positioned at the cell apex and associates with an enigmatic apical vesicle (AV), which docks one or two rhoptries at the site of exocytosis. The interplay among the rhoptries, the AV, and the parasite plasma membrane for secretion remains unclear. Moreover, it is unknown if a similar machinery exists in the deadly malaria parasite Plasmodium falciparum. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct AV docking the tips of the two rhoptries to the RSA. We also discover two new organizations: one in which the AV is absent with one of the two rhoptry tips docks directly to the RSA, and a second in which the two rhoptries fuse together and the common tip docks directly to the RSA. Interestingly, rhoptries among the three states show no significant difference in luminal volume and density, suggesting that the exocytosis of rhoptry contents has not yet occurred, and that these different organizations likely represent sequential states leading to secretion. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to each state, including the opening of a gate-like density in the rhoptry-fused state. These conformational changes of the RSA uncover structural details of a priming process for major rhoptry secretion, which likely occur after initial interaction with a red blood cell. Our results highlight a previously unknown step in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

scholarly journals The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

2020 ◽  
Author(s):  
Brittany L. Carroll ◽  
Tatsuro Nishikino ◽  
Wangbiao Guo ◽  
Shiwei Zhu ◽  
Seiji Kojima ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Direct Evidence ◽  
Electron Tomography ◽  
Vibrio Alginolyticus ◽  
Flagellar Motor ◽  
Large Rearrangement ◽  
Structural Remodeling ◽  
Cryo Electron Tomography ◽  
Ring Structures ◽  
Bacterial Flagellar Motor

ABSTRACTThe bacterial flagellar motor is an intricate nanomachine that switches rotational directions between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants (CCW-biased fliG-G214S and CW-locked fliG-G215A) in Vibrio alginolyticus. Sub-tomogram averaging was utilized to resolve two distinct conformations of the C-ring. Comparison of the C-ring structures in two rotational senses provide direct evidence that the C-ring undergoes major structural remodeling during rotational switch. Specifically, FliG conformational changes elicit a large rearrangement of the C-ring that coincides with rotational switching, whereas FliM and FliN form a spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

scholarly journals The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Brittany L Carroll ◽  
Tatsuro Nishikino ◽  
Wangbiao Guo ◽  
Shiwei Zhu ◽  
Seiji Kojima ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Protein Composition ◽  
Vibrio Alginolyticus ◽  
Flagellar Motor ◽  
Conformational Rearrangement ◽  
Structural Remodeling ◽  
Cryo Electron Tomography ◽  
Bacterial Flagellar Motor ◽  
Rotational Direction

The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

scholarly journals Molecular architecture of black widow spider neurotoxins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Minghao Chen ◽  
Daniel Blum ◽  
Lena Engelhard ◽  
Stefan Raunser ◽  
Richard Wagner ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Calcium Flux ◽  
Membrane Insertion ◽  
Molecular Architecture ◽  
Cyclic Mechanism ◽  
High Resolution Structure ◽  
Specific Receptors ◽  
Β Sheet ◽  
Widow Spider ◽  
Resolution Structure

AbstractLatrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

scholarly journals Molecular architecture of black widow spider neurotoxins

2021 ◽  
Author(s):  
Minghao Chen ◽  
Daniel Blum ◽  
Lena Engelhard ◽  
Stefan Raunser ◽  
Richard Wagner ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Calcium Flux ◽  
Membrane Insertion ◽  
Molecular Architecture ◽  
Cyclic Mechanism ◽  
High Resolution Structure ◽  
Specific Receptors ◽  
Β Sheet ◽  
Widow Spider ◽  
Resolution Structure

AbstractLatrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ɛ-latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear.Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

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