scholarly journals Peptide-Conjugation Induced Conformational Changes in Human IgG1 Observed by Optimized Negative-Staining and Individual-Particle Electron Tomography

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Huimin Tong ◽  
Lei Zhang ◽  
Allan Kaspar ◽  
Matthew J. Rames ◽  
Liqing Huang ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Individual Particle ◽  
Negative Staining ◽  
Peptide Conjugation ◽  
Human Igg1

scholarly journals Nuclear pores constrict upon energy depletion

2020 ◽  
Author(s):  
Christian E Zimmerli ◽  
Matteo Allegretti ◽  
Vasileios Rantos ◽  
Sara K Goetz ◽  
Agnieszka Obarska-Kosinska ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Electron Tomography ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Energy Status ◽  
Energy Depletion ◽  
Time Variations ◽  
Subtomogram Averaging ◽  
Pore Complexes

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins that form an intricate cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter were reported, but the physiological circumstances and the molecular details remain unknown. Here we combined cryo-electron tomography and subtomogram averaging with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. While actively transporting NPCs adopt a dilated conformation, they strongly constrict upon cellular energy depletion. Fluorescence recovery after photo bleaching experiments show that NPC constriction is concomitant with reduced diffusion and active transport across the nuclear envelope. Our data point to a model where the energy status of cells is linked to the conformation of NPC architecture.

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 Microtubule structure by cryo-EM: snapshots of dynamic instability

2018 ◽  
Vol 62 (6) ◽  
pp. 737-751 ◽  
Author(s):  
Szymon W. Manka ◽  
Carolyn A. Moores
Keyword(s):  
Conformational Changes ◽  
Structural Model ◽  
Dynamic Instability ◽  
Electron Tomography ◽  
3D Structure ◽  
Cellular Functions ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Binding Partners ◽  
Gtpase Cycle

The development of cryo-electron microscopy (cryo-EM) allowed microtubules to be captured in their solution-like state, enabling decades of insight into their dynamic mechanisms and interactions with binding partners. Cryo-EM micrographs provide 2D visualization of microtubules, and these 2D images can also be used to reconstruct the 3D structure of the polymer and any associated binding partners. In this way, the binding sites for numerous components of the microtubule cytoskeleton—including motor domains from many kinesin motors, and the microtubule-binding domains of dynein motors and an expanding collection of microtubule associated proteins—have been determined. The effects of various microtubule-binding drugs have also been studied. High-resolution cryo-EM structures have also been used to probe the molecular basis of microtubule dynamic instability, driven by the GTPase activity of β-tubulin. These studies have shown the conformational changes in lattice-confined tubulin dimers in response to steps in the tubulin GTPase cycle, most notably lattice compaction at the longitudinal inter-dimer interface. Although work is ongoing to define a complete structural model of dynamic instability, attention has focused on the role of gradual destabilization of lateral contacts between tubulin protofilaments, particularly at the microtubule seam. Furthermore, lower resolution cryo-electron tomography 3D structures are shedding light on the heterogeneity of microtubule ends and how their 3D organization contributes to dynamic instability. The snapshots of these polymers captured using cryo-EM will continue to provide critical insights into their dynamics, interactions with cellular components, and the way microtubules contribute to cellular functions in diverse physiological contexts.

scholarly journals Single-Molecule 3D Images of “Hole-Hole” IgG1 Homodimers by Individual-Particle Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dongsheng Lei ◽  
Jianfang Liu ◽  
Hongbin Liu ◽  
Thomas E. Cleveland ◽  
John P. Marino ◽  
...  
Keyword(s):  
Single Molecule ◽  
Electron Tomography ◽  
Individual Particle ◽  
3D Images

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 Alteration of Hepatitis A Virus (HAV) Particles by a Soluble Form of HAV Cellular Receptor 1 Containing the Immunoglobulin- and Mucin-Like Regions

2003 ◽  
Vol 77 (16) ◽  
pp. 8765-8774 ◽  
Author(s):  
Erica Silberstein ◽  
Li Xing ◽  
Willem van de Beek ◽  
Jinhua Lu ◽  
Holland Cheng ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Soluble Form ◽  
Pcr Analysis ◽  
Cellular Receptor ◽  
African Green Monkey Kidney ◽  
Negative Stain ◽  
Electron Microscopy Analysis ◽  
Human Igg1

ABSTRACT Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1). The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobulin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobulin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100- to 125S particles. Negative-stain electron microscopy analysis revealed that the 100- to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100- to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating.

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