scholarly journals Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Hans Moldenhauer ◽  
Ignacio Díaz-Franulic ◽  
Horacio Poblete ◽  
David Naranjo
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Tight Binding ◽  
External Solution ◽  
Scorpion Venom ◽  
K Channels ◽  
Interaction Interface ◽  
Dynamic Stochastic

In silico and in vitro studies have made progress in understanding protein–protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein–protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

scholarly journals Visualization of Calcium Ion Loss from Rotavirus during Cell Entry

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Eric N. Salgado ◽  
Brian Garcia Rodriguez ◽  
Nagarjun Narayanaswamy ◽  
Yamuna Krishnan ◽  
Stephen C. Harrison
Keyword(s):  
Conformational Changes ◽  
Outer Layer ◽  
Live Cell Imaging ◽  
Molecular Mechanisms ◽  
Cell Imaging ◽  
Tight Binding ◽  
Live Cell ◽  
Membrane Disruption ◽  
Molecular Events

ABSTRACTBound calcium ions stabilize many nonenveloped virions. Loss of Ca2+from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca2+-stabilized protein (VP7). Membrane-coupled conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double-layered inner capsid particle (DLP) into the cytosol. Removal of Ca2+in vitrostrips away both outer layer proteins; we and others have postulated that the loss of Ca2+triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca2+sensor, the timing of Ca2+loss from entering virions with respect to the dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer layer dissociation and DLP release. The Ca2+sensor, placed on VP5*, monitors the Ca2+concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1-min duration) loss of Ca2+precedes the onset of VP7 dissociation by about 2 min and DLP release by about 7 min. Coupled with our previous results showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca2+loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry.IMPORTANCENonenveloped viruses penetrate into the cytosol of the cells that they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying nonenveloped virus entry. We report here how the use of a fluorescent Ca2+sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca2+loss from an entering particle and other molecular events in the entry pathway. Specific Ca2+binding stabilizes many other viruses of eukaryotes, and Ca2+loss appears to be a trigger for steps in penetration or uncoating. The experimental design that we describe may be useful for studying entry of other viral pathogens.

scholarly journals Conformational heterogeneity of the AAA ATPase p97 characterized by single particle cryo-EM

2014 ◽  
Vol 70 (a1) ◽  
pp. C853-C853
Author(s):  
Driss Mountassif ◽  
Lucien Fabre ◽  
Kaustuv Basu ◽  
Mihnea Bostina ◽  
Slavica Jonic ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Single Particle ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Normal Mode Analysis ◽  
Single Amino Acid ◽  
Mode Analysis ◽  
Conformational Heterogeneity ◽  
Aaa Atpase

p97, a member of the AAA (ATPase Associated with various Activities) ATPase family, is essential and centrally involved in a wide variety of cellular processes. Single amino-acid substitutions in p97 have been associated with the severe degenerative disorder of Inclusion Body Myopathy associated with Paget disease of bone and Frontotemporal Dementia (IBMPFD) as well as amytropic leteral sclerosis (ALS). Current models propose that p97 acts as a motor transmitting the energy from the ATPase cycle to conformational changes of substrate protein complexes causing segregation, remodeling or translocation. Mutations at the interface between the N and the D1 domains impact the ATPase activity and the conformation of D2 on the opposite side of the protein complex, suggesting intermolecular communication. Because of limited structural information, the molecular mechanisms on how p97 drives its activities and the molecular basis for transmission of information within the molecule remain elusive. Structural heterogeneity is observed in vitro and is likely relevant for the in vivo biological function of p97. Single particle cryo-EM is the method of choice to study a flexible complex. The technique allows study in solution and also deals with sample heterogeneity by image classification. We have set-up the characterization of the conformational heterogeneity in WT and disease relevant p97 mutant using multi-likelihood classification and Hybrid Electron Microscopy Normal Mode Analysis HEMNMA. The multi-likelihood analysis shows a link between the conformations of the N and D2 domains. HEMNMA allows the analysis of the asymmetry of the conformational changes. Together these studies describe the structural flexibility of p97 and the coupling of the ATPase activity with conformational changes in health and in disease. Study of this model system also allows the development of new methods to understand the conformational heterogeneity of other protein complexes.

scholarly journals Novel Escherichia coli RNA Polymerase Binding Protein Encoded by Bacteriophage T5

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 807
Author(s):  
Evgeny Klimuk ◽  
Vladimir Mekler ◽  
Darya Lavysh ◽  
Marina Serebryakova ◽  
Natalia Akulenko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Molecular Mechanisms ◽  
Tight Binding ◽  
Virus Particles ◽  
Regulatory Cascade ◽  
Infected Cells ◽  
Polymerase Binding ◽  
Bacteriophage T5

The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the σ70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP β subunit and moderately inhibits RNAP interaction with the discriminator region of σ70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription.

scholarly journals Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation

2020 ◽  
Vol 21 (12) ◽  
pp. 4421
Author(s):  
Yurii S. Borovikov ◽  
Armen O. Simonyan ◽  
Stanislava V. Avrova ◽  
Vladimir V. Sirenko ◽  
Charles S. Redwood ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Muscle Weakness ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Spatial Arrangement ◽  
Muscle Fibres ◽  
Thin Filaments ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Substitution of Ala for Glu residue in position 173 of γ-tropomyosin (Tpm3.12) is associated with muscle weakness. Here we observe that this mutation increases myofilament Ca2+-sensitivity and inhibits in vitro actin-activated ATPase activity of myosin subfragment-1 at high Ca2+. In order to determine the critical conformational changes in myosin, actin and tropomyosin caused by the mutation, we used the technique of polarized fluorimetry. It was found that this mutation changes the spatial arrangement of actin monomers and myosin heads, and the position of the mutant tropomyosin on the thin filaments in muscle fibres at various mimicked stages of the ATPase cycle. At low Ca2+ the E173A mutant tropomyosin shifts towards the inner domains of actin at all stages of the cycle, and this is accompanied by an increase in the number of switched-on actin monomers and myosin heads strongly bound to F-actin even at relaxation. Contrarily, at high Ca2+ the amount of the strongly bound myosin heads slightly decreases. These changes in the balance of the strongly bound myosin heads in the ATPase cycle may underlie the occurrence of muscle weakness. W7, an inhibitor of troponin Ca2+-sensitivity, restores the increase in the number of myosin heads strongly bound to F-actin at high Ca2+ and stops their strong binding at relaxation, suggesting the possibility of using Ca2+-desensitizers to reduce the damaging effect of the E173A mutation on muscle fibre contractility.

scholarly journals A conceptual view at microtubule plus end dynamics in neuronal axons

2016 ◽  
Author(s):  
André Voelzmann ◽  
Ines Hahn ◽  
Simon P. Pearce ◽  
Natalia Sánchez-Soriano ◽  
Andreas Prokop
Keyword(s):  
Nervous System ◽  
Conceptual Understanding ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Building Blocks ◽  
Cellular Level ◽  
Cellular Functions ◽  
Dynamic Changes ◽  
Plastic Rearrangement

AbstractAxons are the cable-like protrusions of neurons which wire up the nervous system. Polar bundles of microtubules (MTs) constitute their structural backbones and are highways for life-sustaining transport between proximal cell bodies and distal synapses. Any morphogenetic changes of axons during development, plastic rearrangement, regeneration or degeneration depend on dynamic changes of these MT bundles. A key mechanism for implementing such changes is the coordinated polymerisation and depolymerisation at the plus ends of MTs within these bundles. To gain an understanding of how such regulation can be achieved at the cellular level, we provide here an integrated overview of the extensive knowledge we have about the molecular mechanisms regulating MT de/polymerisation. We first summarise insights gained from work in vitro, then describe the machinery which supplies the essential tubulin building blocks, the protein complexes associating with MT plus ends, and MT shaft-based mechanisms that influence plus end dynamics. We briefly summarise the contribution of MT plus end dynamics to important cellular functions in axons, and conclude by discussing the challenges and potential strategies of integrating the existing molecular knowledge into conceptual understanding at the level of axons.

scholarly journals Hbo1 Links p53-Dependent Stress Signaling to DNA Replication Licensing

2007 ◽  
Vol 28 (1) ◽  
pp. 140-153 ◽  
Author(s):  
Masayoshi Iizuka ◽  
Olga F. Sarmento ◽  
Takao Sekiya ◽  
Heidi Scrable ◽  
C. David Allis ◽  
...  
Keyword(s):  
Dna Replication ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Stress Signaling ◽  
Dependent Manner ◽  
Histone H4 ◽  
Steroid Dependent ◽  
Histone H4 Acetylation ◽  
Replication Fork Arrest

ABSTRACT Hbo1 is a histone acetyltransferase (HAT) that is required for global histone H4 acetylation, steroid-dependent transcription, and chromatin loading of MCM2-7 during DNA replication licensing. It is the catalytic subunit of protein complexes that include ING and JADE proteins, growth regulatory factors and candidate tumor suppressors. These complexes are thought to act via tumor suppressor p53, but the molecular mechanisms and links between stress signaling and chromatin, are currently unknown. Here, we show that p53 physically interacts with Hbo1 and negatively regulates its HAT activity in vitro and in cells. Two physiological stresses that stabilize p53, hyperosmotic shock and DNA replication fork arrest, also inhibit Hbo1 HAT activity in a p53-dependent manner. Hyperosmotic stress during G1 phase specifically inhibits the loading of the MCM2-7 complex, providing an example of the chromatin output of this pathway. These results reveal a direct regulatory connection between p53-responsive stress signaling and Hbo1-dependent chromatin pathways.

scholarly journals Dynamin-2 R465W mutation induces long range perturbation in highly ordered oligomeric structures

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Fernando Hinostroza ◽  
Alan Neely ◽  
Ingrid Araya-Duran ◽  
Vanessa Marabolí ◽  
Jonathan Canan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Function ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Coarse Grained ◽  
Cell Physiology ◽  
Missense Mutations ◽  
Small Change ◽  
Dynamics Simulations ◽  
Dynamin 2

Abstract High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.

scholarly journals Mechanism of gasdermin D recognition by inflammatory caspases and their inhibition by a gasdermin D-derived peptide inhibitor

2018 ◽  
Vol 115 (26) ◽  
pp. 6792-6797 ◽  
Author(s):  
Jie Yang ◽  
Zhonghua Liu ◽  
Chuanping Wang ◽  
Rui Yang ◽  
Joseph K. Rathkey ◽  
...  
Keyword(s):  
Cell Death ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Membrane Lipids ◽  
Enzyme Inhibitor ◽  
Apoptotic Cell ◽  
Specific Inhibitor ◽  
Caspase 1 ◽  
Inflammatory Caspases

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor,N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1β release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme–inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.

scholarly journals Neuronal Glycoprotein M6a: An Emerging Molecule in Chemical Synapse Formation and Dysfunction

2021 ◽  
Vol 13 ◽  
Author(s):  
Antonella León ◽  
Gabriela I. Aparicio ◽  
Camila Scorticati
Keyword(s):  
Large Scale ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
Synapse Formation ◽  
Disease Risk ◽  
Protein Complexes ◽  
Synaptic Function ◽  
Chemical Synapse ◽  
Gene Target

The cellular and molecular mechanisms underlying neuropsychiatric and neurodevelopmental disorders show that most of them can be categorized as synaptopathies—or damage of synaptic function and plasticity. Synaptic formation and maintenance are orchestrated by protein complexes that are in turn regulated in space and time during neuronal development allowing synaptic plasticity. However, the exact mechanisms by which these processes are managed remain unknown. Large-scale genomic and proteomic projects led to the discovery of new molecules and their associated variants as disease risk factors. Neuronal glycoprotein M6a, encoded by the GPM6A gene is emerging as one of these molecules. M6a has been involved in neuron development and synapse formation and plasticity, and was also recently proposed as a gene-target in various neuropsychiatric disorders where it could also be used as a biomarker. In this review, we provide an overview of the structure and molecular mechanisms by which glycoprotein M6a participates in synapse formation and maintenance. We also review evidence collected from patients carrying mutations in the GPM6A gene; animal models, and in vitro studies that together emphasize the relevance of M6a, particularly in synapses and in neurological conditions.

scholarly journals In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

2022 ◽  
Author(s):  
Robert Fagiewicz ◽  
Corinne Crucifix ◽  
Celia Deville ◽  
Bruno Kieffer ◽  
Yves Nomine ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Complexes ◽  
Cytoplasmic Dynein ◽  
Full Length ◽  
Recombinant Production ◽  
In Vitro Characterization ◽  
Microtubule Motor ◽  
Glial Progenitor Cells ◽  
Development In Vitro

The cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is one of the most prominent examples within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis as well as interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2 and cytoplasmic dynein-1. We also describe pH-dependent conformational changes of BicD2 using cryoEM, template-free structure predictions, and biophysical tools. Our results will help defining the biochemical parameters for the invitro reconstitution of higher order BicD2 protein complexes.

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