Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations

AbstractTo enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.

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A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

Science Advances ◽

10.1126/sciadv.abc6345 ◽

2021 ◽

Vol 7 (13) ◽

pp. eabc6345

Author(s):

Shrawan Kumar Mageswaran ◽

Wei Yuan Yang ◽

Yogaditya Chakrabarty ◽

Catherine M. Oikonomou ◽

Grant J. Jensen

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Electron Tomography ◽

Membrane Damage ◽

Light And Electron Microscopy ◽

Actin Dynamics ◽

Endosomal Sorting ◽

Plasma Membrane Damage ◽

Cryo Electron Tomography ◽

Vacuolar Protein

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

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CryoEM and computer simulations reveal a novel kinase conformational switch in bacterial chemotaxis signaling

eLife ◽

10.7554/elife.08419 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 67

Author(s):

C Keith Cassidy ◽

Benjamin A Himes ◽

Frances J Alvarez ◽

Jun Ma ◽

Gongpu Zhao ◽

...

Keyword(s):

Electron Tomography ◽

Conformational Dynamics ◽

Bacterial Chemotaxis ◽

Adaptor Protein ◽

Structural Description ◽

Structural Constraints ◽

The Core ◽

Molecular Events ◽

Density Map ◽

Dynamics Simulations

Chemotactic responses in bacteria require large, highly ordered arrays of sensory proteins to mediate the signal transduction that ultimately controls cell motility. A mechanistic understanding of the molecular events underlying signaling, however, has been hampered by the lack of a high-resolution structural description of the extended array. Here, we report a novel reconstitution of the array, involving the receptor signaling domain, histidine kinase CheA, and adaptor protein CheW, as well as a density map of the core-signaling unit at 11.3 Å resolution, obtained by cryo-electron tomography and sub-tomogram averaging. Extracting key structural constraints from our density map, we computationally construct and refine an atomic model of the core array structure, exposing novel interfaces between the component proteins. Using all-atom molecular dynamics simulations, we further reveal a distinctive conformational change in CheA. Mutagenesis and chemical cross-linking experiments confirm the importance of the conformational dynamics of CheA for chemotactic function.

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The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling During Infection

Science ◽

10.1126/science.1231887 ◽

2013 ◽

Vol 339 (6119) ◽

pp. 576-579 ◽

Cited By ~ 134

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.

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Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

Journal of Bacteriology ◽

10.1128/jb.00695-16 ◽

2016 ◽

Vol 199 (3) ◽

Cited By ~ 28

Author(s):

Zhuan Qin ◽

Wei-ting Lin ◽

Shiwei Zhu ◽

Aime T. Franco ◽

Jun Liu

Keyword(s):

Helicobacter Pylori ◽

Molecular Mechanisms ◽

Electron Tomography ◽

Bacterial Pathogen ◽

Human Stomach ◽

Content Type ◽

Flagellar Motors ◽

Cryo Electron Tomography ◽

Flagellar Assembly ◽

H Pylori

ABSTRACT Helicobacter pylori is a bacterial pathogen that can cause many gastrointestinal diseases, including ulcers and gastric cancer. A unique chemotaxis-mediated motility is critical for H. pylori to colonize in the human stomach and to establish chronic infection, but the underlying molecular mechanisms are not well understood. Here, we employ cryo-electron tomography (cryo-ET) to reveal detailed structures of the H. pylori cell envelope, including the sheathed flagella and chemotaxis arrays. Notably, H. pylori possesses a distinctive periplasmic cage-like structure with 18-fold symmetry. We propose that this structure forms a robust platform for recruiting 18 torque generators, which likely provide the higher torque needed for swimming in high-viscosity environments. We also reveal a series of key flagellar assembly intermediates, providing structural evidence that flagellar assembly is tightly coupled with the biogenesis of the membrane sheath. Finally, we determine the structure of putative chemotaxis arrays at the flagellar pole, which have implications for how the direction of flagellar rotation is regulated. Together, our pilot cryo-ET studies provide novel structural insights into the unipolar flagella of H. pylori and lay a foundation for a better understanding of the unique motility of this organism. IMPORTANCE Helicobacter pylori is a highly motile bacterial pathogen that colonizes approximately 50% of the world's population. H. pylori can move readily within the viscous mucosal layer of the stomach. It has become increasingly clear that its unique flagella-driven motility is essential for successful gastric colonization and pathogenesis. Here, we use advanced imaging techniques to visualize novel in situ structures with unprecedented detail in intact H. pylori cells. Remarkably, H. pylori possesses multiple unipolar flagella, which are driven by one of the largest flagellar motors found in bacteria. These large motors presumably provide the higher torque needed by the bacterial pathogens to navigate in the viscous environment of the human stomach.

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Comparison of the envelope architecture of E. coli using two methods: CEMOVIS and cryo-electron tomography

Journal of Electron Microscopy ◽

10.1093/jmicro/dfq056 ◽

2010 ◽

Vol 59 (5) ◽

pp. 419-426 ◽

Cited By ~ 14

Author(s):

A. Kishimoto-Okada ◽

S. Murakami ◽

Y. Ito ◽

N. Horii ◽

H. Furukawa ◽

...

Keyword(s):

Electron Tomography ◽

E Coli ◽

Cryo Electron Tomography

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Alternative Architecture of the E. coli Chemosensory Array

Biomolecules ◽

10.3390/biom11040495 ◽

2021 ◽

Vol 11 (4) ◽

pp. 495

Author(s):

Alister Burt ◽

C. Keith Cassidy ◽

Phillip J. Stansfeld ◽

Irina Gutsche

Keyword(s):

Electron Tomography ◽

Structural Basis ◽

Published Data ◽

Molecular Models ◽

E Coli ◽

Array Architecture ◽

Tremendous Progress ◽

Cryo Electron Tomography ◽

Functional Implications ◽

And Function

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

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Alternative Architecture of the E. Coli Chemosensory Array

10.21203/rs.3.rs-124318/v1 ◽

2020 ◽

Author(s):

Alister Burt ◽

C Cassidy ◽

Phillip Stansfeld ◽

Irina Gutsche

Keyword(s):

Electron Tomography ◽

Structural Basis ◽

Published Data ◽

Molecular Models ◽

E Coli ◽

Array Architecture ◽

Tremendous Progress ◽

Cryo Electron Tomography ◽

Functional Implications ◽

And Function

Abstract Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

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Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

10.1101/773416 ◽

2019 ◽

Cited By ~ 2

Author(s):

Gang Fu ◽

Lei Zhao ◽

Erin Dymek ◽

Yuqing Hou ◽

Kangkang Song ◽

...

Keyword(s):

Molecular Mechanisms ◽

Electron Tomography ◽

3D Structure ◽

Three Dimensional ◽

Protein Composition ◽

Wild Type ◽

Ciliary Motility ◽

Cryo Electron Tomography ◽

Central Apparatus ◽

Subunit Organization

AbstractNearly all motile cilia contain a central apparatus (CA) composed of two connected singlet-microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild type Chlamydomonas with CA mutants. We identified a large (>2 MDa) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold-labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and three-dimensional (3D) structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.SummaryFu et al. use a wild-type vs. mutant comparison and cryo-electron tomography of Chlamydomonas flagella to identify central apparatus (CA) subunits and visualize their location in the native 3D CA structure. The study provides a better understanding of the CA and how it regulates ciliary motility.

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Alternative architecture of the E. coli chemosensory array

10.1101/2021.02.05.429929 ◽

2021 ◽

Author(s):

Alister Burt ◽

C. Keith Cassidy ◽

Phillip J. Stansfeld ◽

Irina Gutsche

Keyword(s):

Electron Tomography ◽

Structural Basis ◽

Published Data ◽

Molecular Models ◽

E Coli ◽

Array Architecture ◽

Tremendous Progress ◽

Cryo Electron Tomography ◽

Functional Implications ◽

And Function

AbstractChemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

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3D-Visualization of Amyloid-β Oligomer and Fibril Interactions with Lipid Membranes by Cryo-Electron Tomography

10.1101/2020.07.21.214072 ◽

2020 ◽

Author(s):

Yao Tian ◽

Ruina Liang ◽

Amit Kumar ◽

Piotr Szwedziak ◽

John H. Viles

Keyword(s):

Lipid Bilayers ◽

Molecular Mechanisms ◽

Lipid Membrane ◽

Electron Tomography ◽

3D Visualization ◽

Membrane Integrity ◽

Amyloid Β ◽

Lipid Vesicles ◽

Aβ Oligomers ◽

Cryo Electron Tomography

ABSTRACTAmyloid-β (Aβ) monomers assemble into mature fibrils via a range of metastable oligomeric and protofibrillar intermediates. These Aβ assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer’s disease. However, molecular mechanisms of Aβ cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aβ assembly types and their interaction with liposomes. Aβ oligomers bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Furthermore, curvilinear protofibrils also insert into the bilayer, orthogonally to the membrane surface. Aβ oligomers concentrate at the interface of vesicles and form a network of Aβ-linked liposomes. While crucially, monomeric and fibrillar Aβ have relatively little impact on the membrane. Changes to lipid membrane composition highlights a significant role for GM1-ganglioside in promoting Aβ-membrane interactions. The different effects of Aβ assembly forms observed align with the highlighted cytotoxicity reported for Aβ oligomers. The wide-scale incorporation of Aβ oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

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