Microtubule structure by cryo-EM: snapshots of dynamic instability

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.

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Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

eLife ◽

10.7554/elife.03680 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 69

Author(s):

Joseph Atherton ◽

Irene Farabella ◽

I-Mei Yu ◽

Steven S Rosenfeld ◽

Anne Houdusse ◽

...

Keyword(s):

Electron Microscopy ◽

Conformational Changes ◽

Catalytic Site ◽

Fundamental Question ◽

Force Generation ◽

Atp Binding ◽

Cellular Functions ◽

Microtubule Binding ◽

Cryo Electron Microscopy ◽

Adp Release

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

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ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2008.0191 ◽

2008 ◽

Vol 364 (1514) ◽

pp. 247-255 ◽

Cited By ~ 32

Author(s):

Daniella Muallem ◽

Paola Vergani

Keyword(s):

Cystic Fibrosis ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Atp Binding ◽

Functional Difference ◽

Transmembrane Conductance Regulator ◽

Cellular Functions ◽

Transmembrane Conductance ◽

Binding Domains ◽

Cystic Fibrosis Transmembrane

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and γ-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

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Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

10.1101/2020.12.20.423706 ◽

2020 ◽

Author(s):

Stefan Niekamp ◽

Nico Stuurman ◽

Nan Zhang ◽

Ronald D. Vale

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

In Silico ◽

Conformational Transition ◽

Conformational Dynamics ◽

Motor Protein ◽

Single Molecule Imaging ◽

Microtubule Binding ◽

Binding Domains ◽

Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule binding domains (MTBD) that step along the track. Here, we simultaneously tracked two MTBDs and one AAA ring of a single dynein, as it undergoes hundreds of steps with nanometer precision using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take different sized steps. This variability in the movement between AAA ring and MTBD results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

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Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

eLife ◽

10.7554/elife.47145 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Samuel E Lacey ◽

Shaoda He ◽

Sjors HW Scheres ◽

Andrew P Carter

Keyword(s):

Conformational Changes ◽

Coiled Coil ◽

Cytoplasmic Dynein ◽

Cross Sectional ◽

Microtubule Binding ◽

High Affinity ◽

Binding Domains ◽

Microtubule Binding Domain ◽

Axonemal Dynein ◽

Cilia And Flagella

Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

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Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2101391118 ◽

2021 ◽

Vol 118 (31) ◽

pp. e2101391118

Author(s):

Stefan Niekamp ◽

Nico Stuurman ◽

Nan Zhang ◽

Ronald D. Vale

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

In Silico ◽

Conformational Transition ◽

Conformational Dynamics ◽

Motor Protein ◽

Single Molecule Imaging ◽

Microtubule Binding ◽

Binding Domains ◽

Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

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Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

10.1101/590786 ◽

2019 ◽

Cited By ~ 1

Author(s):

Samuel E. Lacey ◽

Shaoda He ◽

Sjors H. W. Scheres ◽

Andrew P. Carter

Keyword(s):

Conformational Changes ◽

Coiled Coil ◽

Cytoplasmic Dynein ◽

Cross Sectional ◽

Microtubule Binding ◽

High Affinity ◽

Binding Domains ◽

Microtubule Binding Domain ◽

Axonemal Dynein ◽

Cilia And Flagella

AbstractDyneins are motor proteins responsible for transport in the cytoplasm and the beating of the axoneme in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a long coiled-coil stalk. Here we address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using the stand-alone Relion package. We show the majority of the MTBD is remarkably rigid upon binding, with the transition to the high affinity state controlled by the movement of a single helix at the MTBD interface. In addition DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that reaches over and contacts the adjacent protofilament. Unexpectedly we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

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Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

Science ◽

10.1126/science.abd4914 ◽

2021 ◽

Vol 371 (6525) ◽

pp. eabd4914

Author(s):

Sudarshan Gadadhar ◽

Gonzalo Alvarez Viar ◽

Jan Niklas Hansen ◽

An Gong ◽

Aleksandr Kostarev ◽

...

Keyword(s):

Posttranslational Modifications ◽

Male Fertility ◽

Electron Tomography ◽

Structural Basis ◽

Cellular Functions ◽

Flagellar Beat ◽

Cryo Electron Tomography ◽

Axonemal Dynein ◽

Dynein Arms ◽

Cilia And Flagella

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

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Molecular Processing of Tau Protein in Progressive Supranuclear Palsy: Neuronal and Glial Degeneration

Journal of Alzheimer s Disease ◽

10.3233/jad-201139 ◽

2021 ◽

Vol 79 (4) ◽

pp. 1517-1531

Author(s):

Alejandra Martínez-Maldonado ◽

Miguel Ángel Ontiveros-Torres ◽

Charles R. Harrington ◽

José Francisco Montiel-Sosa ◽

Raúl García-Tapia Prandiz ◽

...

Keyword(s):

Progressive Supranuclear Palsy ◽

Glial Cells ◽

Conformational Changes ◽

Tau Protein ◽

Paired Helical Filaments ◽

Phosphorylated Tau ◽

Clinical Heterogeneity ◽

Post Translational Modifications ◽

Binding Domains ◽

Tau Isoforms

Background: Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP) are examples of neurodegenerative diseases, characterized by abnormal tau inclusions, that are called tauopathies. AD is characterized by highly insoluble paired helical filaments (PHFs) composed of tau with abnormal post-translational modifications. PSP is a neurodegenerative disease with pathological and clinical heterogeneity. There are six tau isoforms expressed in the adult human brain, with repeated microtubule-binding domains of three (3R) or four (4R) repeats. In AD, the 4R:3R ratio is 1:1. In PSP, the 4R isoform predominates. The lesions in PSP brains contain phosphorylated tau aggregates in both neurons and glial cells. Objective: Our objective was to evaluate and compare the processing of pathological tau in PSP and AD. Methods: Double and triple immunofluorescent labeling with antibodies to specific post-translational tau modifications (phosphorylation, truncation, and conformational changes) and thiazin red (TR) staining were carried out and analyzed by confocal microscopy. Results: Our results showed that PSP was characterized by phosphorylated tau in neurofibrillary tangles (NFTs) and glial cells. Tau truncated at either Glu391 or Asp421 was not observed. Extracellular NFTs (eNFTs) and glial cells in PSP exhibited a strong affinity for TR in the absence of intact or phosphorylated tau. Conclusion: Phosphorylated tau was as abundant in PSP as in AD. The development of eNFTs from both glial cells and neuronal bodies suggests that truncated tau species, different from those observed in AD, could be present in PSP. Additional studies on truncated tau within PSP lesions could improve our understanding of the pathological processing of tau and help identify a discriminatory biomarker for AD and PSP.

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A structural framework for unidirectional transport by a bacterial ABC exporter

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006526117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19228-19236

Author(s):

Chengcheng Fan ◽

Jens T. Kaiser ◽

Douglas C. Rees

Keyword(s):

Conformational Changes ◽

Iron Homeostasis ◽

Atp Hydrolysis ◽

Transmembrane Helix ◽

Reverse Direction ◽

Conformational States ◽

X Ray Crystallography ◽

Binding Domains ◽

Structural Framework ◽

Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

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New insights into the structural basis of integrin activation

Blood ◽

10.1182/blood-2003-01-0334 ◽

2003 ◽

Vol 102 (4) ◽

pp. 1155-1159 ◽

Cited By ~ 133

Author(s):

Jian-Ping Xiong ◽

Thilo Stehle ◽

Simon L. Goodman ◽

M. Amin Arnaout

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Structural Basis ◽

Integrin Activation ◽

Cellular Functions ◽

Electron Microscopic ◽

The Past ◽

Intracellular Signals ◽

Bidirectional Signaling ◽

New Hypothesis

Abstract Integrins are cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane and thus influence most cellular functions. Intracellular signals switch integrins into a ligand-competent state as a result of elicited conformational changes in the integrin ectodomain. Binding of extracellular ligands induces, in turn, structural changes that convey distinct signals to the cell interior. The structural basis of this bidirectional signaling has been the focus of intensive study for the past 3 decades. In this perspective, we develop a new hypothesis for integrin activation based on recent crystallographic, electron microscopic, and biochemical studies.

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