Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM.

ABSTRACT: Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

SUBMITTER: Kubo S

PROVIDER: S-EPMC8419702 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM.

Kubo Shintaroh S Yang Shun Kai SK Black Corbin S CS Dai Daniel D Valente-Paterno Melissa M Gaertig Jacek J Ichikawa Muneyoshi M Bui Khanh Huy KH

EMBO reports 20210802 9

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA com ...[more]

PMID: 34338432

Similar Datasets

Cryo-EM shows how dynactin recruits two dyneins for faster movement.

Project description:Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a dynein tail-dynactin-BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein-dynactin transport machine.

| S-EPMC5988349 | biostudies-literature

Structural mechanisms of inflammasome regulation revealed by cryo-EM studies.

Project description:Inflammasomes are cytosolic protein complexes that form in response to pathogen or damage signals and initiate inflammation. Signal transduction in the inflammasome pathway occurs via protein-protein interaction, protein conformational change, and oligomerization. Recent advances in structural biology have provided multiple insights in inflammasome regulation that are both biologically intriguing and therapeutically valuable. In this review, we summarize the current understanding of three most studied inflammasome complexes: the NAIP/NLRC4, NLRP1, and NLRP3 inflammasomes. We discuss the general mechanisms and unique features of their regulation and how investigating these systems may contribute to therapeutic applications.

| S-EPMC9597862 | biostudies-literature

Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM.

Project description:Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+ binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.

| S-EPMC6414200 | biostudies-literature

Autoinhibition and activation of myosin VI revealed by its cryo-EM structure.

Project description:Myosin VI is the only molecular motor that moves towards the minus end along actin filaments. Numerous cellular processes require myosin VI and tight regulations of the motor's activity. Defects in myosin VI activity are known to cause genetic diseases such as deafness and cardiomyopathy. However, the molecular mechanisms underlying the activity regulation of myosin VI remain elusive. Here, we determined the high-resolution cryo-electron microscopic structure of myosin VI in its autoinhibited state. Our structure reveals that autoinhibited myosin VI adopts a compact, monomeric conformation via extensive interactions between the head and tail domains, orchestrated by an elongated single-α-helix region resembling a "spine". This autoinhibited structure effectively blocks cargo binding sites and represses the motor's ATPase activity. Certain cargo adaptors such as GIPC can release multiple inhibitory interactions and promote motor activity, pointing to a cargo-mediated activation of the processive motor. Moreover, our structural findings allow rationalization of disease-associated mutations in myosin VI. Beyond the activity regulation mechanisms of myosin VI, our study also sheds lights on how activities of other myosin motors such as myosin VII and X might be regulated.

| S-EPMC10853514 | biostudies-literature

Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates.

Project description:Outer arm dynein (OAD) is the main force generator of ciliary beating. Although OAD loss is the most frequent cause of human primary ciliary dyskinesia, the docking mechanism of OAD onto the ciliary doublet microtubule (DMT) remains elusive in vertebrates. Here, we analyzed the functions of Calaxin/Efcab1 and Armc4, the two of five components of vertebrate OAD-DC (docking complex), using zebrafish spermatozoa and cryo-electron tomography. Mutation of armc4 caused complete loss of OAD, whereas mutation of calaxin caused only partial loss of OAD. Detailed structural analysis revealed that calaxin-/- OADs are tethered to DMT through DC components other than Calaxin, and that recombinant Calaxin can autonomously rescue the deficient DC structure and the OAD instability. Our data demonstrate the discrete roles of Calaxin and Armc4 in the OAD-DMT interaction, suggesting the stabilizing process of OAD docking onto DMT in vertebrates.

| S-EPMC10139691 | biostudies-literature

Two-headed outer- and inner-arm dyneins of Leishmania sp bear conserved IQ-like motifs.

Project description:Dyneins are high molecular weight microtubule based motor proteins responsible for beating of the flagellum. The flagellum is important for the viability of trypanosomes like Leishmania. However, very little is known about dynein and its role in flagellar motility in such trypanosomatid species. Here, we have identified genes in five species of Leishmania that code for outer-arm dynein (OAD) heavy chains α and β, and inner-arm dynein (IAD) heavy chains 1α and 1β using BLAST and MSA. Our sequence analysis indicates that unlike the three-headed outer-arm dyneins of Chlamydomonas and Tetrahymena, the outer-arm dyneins of the genus Leishmania are two-headed, lacking the γ chain like that of metazoans. N-terminal sequence analysis revealed a conserved IQ-like calmodulin binding motif in the outer-arm α and inner-arm 1α dynein heavy chain in the five species of Leishmania similar to Chlamydomonas reinhardtii outer-arm γ. It was predicted that both motifs were incapable of binding calmodulin. Phosphorylation site prediction revealed conserved serine and threonine residues in outer-arm dynein α and inner-arm 1α as putative phosphorylation sites exclusive to Leishmania but not in Trypanosoma brucei suggesting that regulation of dynein activity might be via phosphorylation of these IQ-like motifs in Leishmania sp.

| S-EPMC5669419 | biostudies-literature

Mechanism of NAIP-NLRC4 inflammasome activation revealed by cryo-EM structure of unliganded NAIP5.

Project description:The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in the host defense against bacterial infection. NAIPs interact with conserved bacterial ligands and activate the NLR family caspase recruitment domain containing protein 4 (NLRC4) to initiate the NAIP-NLRC4 inflammasome pathway. Here we found the process of NAIP activation is completely different from NLRC4. Our cryo-EM structure of unliganded mouse NAIP5 adopts an unprecedented wide-open conformation, with the nucleating surface fully exposed and accessible to recruit inactive NLRC4. Upon ligand binding, the winged helix domain (WHD) of NAIP5 undergoes roughly 20° rotation to form a steric clash with the inactive NLRC4, which triggers the conformational change of NLRC4 from inactive to active state. We also show the rotation of WHD places the 17-18 loop at a position that directly bind the active NLRC4 and stabilize the NAIP5-NLRC4 complex. Overall, these data provide structural mechanisms of inactive NAIP5, the process of NAIP5 activation and NAIP-dependent NLRC4 activation.

| S-EPMC10576962 | biostudies-literature

Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM.

Project description:Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre2-loxP (110 kDa), and tetrameric Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.

| S-EPMC8860596 | biostudies-literature

Cryo-EM structures of human ClpXP reveal mechanisms of assembly and proteolytic activation.

Project description:The human ClpXP complex (hClpXP) plays a central role in mitochondrial protein quality control by degrading misfolded or unneeded proteins. While bacterial ClpXP complexes have been extensively characterized, the molecular determinants underlying hClpXP assembly and regulation are not as well understood. We determined cryo-electron microscopy (cryo-EM) structures of hClpP in isolation and in complex with hClpX, revealing how hClpX binding promotes rearrangement of an asymmetric hClpP heptamer to assemble as a symmetric tetradecamer. Our hClpXP structure also highlights the stabilizing role of a previously uncharacterized eukaryotic ClpX sequence, referred to as the E-loop, and its importance in ATPase activity and hexamer assembly. We further show that peptide interaction with the hClpP proteolytic active site promotes the complex to adopt a proteolytically competent conformation. Together, these findings advance our understanding of the molecular mechanisms defining hClpXP activation and function.

| S-EPMC11601447 | biostudies-literature

Coma-corrected rapid single-particle cryo-EM data collection on the CRYO ARM 300.

Project description:Single-particle cryogenic electron microscopy has recently become a major method for determining the structures of proteins and protein complexes. This has markedly increased the demand for throughput of high-resolution electron microscopes, which are required to produce high-resolution images at high rates. An increase in data-collection throughput can be achieved by using large beam-image shifts combined with off-axis coma correction, enabling the acquisition of multiple images from a large area of the EM grid without moving the microscope stage. Here, the optical properties of the JEOL CRYO ARM 300 electron microscope equipped with a K3 camera were characterized under off-axis illumination conditions. It is shown that efficient coma correction can be achieved for beam-image shifts with an amplitude of at least 10 µm, enabling a routine throughput for data collection of between 6000 and 9000 images per day. Use of the benchmark for the rapid data-collection procedure (with beam-image shifts of up to 7 µm) on apoferritin resulted in a reconstruction at a resolution of 1.7 Å. This demonstrates that the rapid automated acquisition of high-resolution micrographs is possible using a CRYO ARM 300.

| S-EPMC8098478 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data