1. Structural Biology and Molecular Biophysics

Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy

  1. Joanna Andrecka
  2. Jaime Ortega Arroyo
  3. Yasuharu Takagi
  4. Gabrielle de Wit
  5. Adam Fineberg
  6. Lachlan MacKinnon
  7. Gavin Young
  8. James R Sellers
  9. Philipp Kukura  Is a corresponding author
  1. University of Oxford, United Kingdom
  2. National Heart, Lung and Blood Institute, National Institutes of Health, United States
https://doi.org/10.7554/eLife.05413
Share this article
Cite this article
  1. Joanna Andrecka
  2. Jaime Ortega Arroyo
  3. Yasuharu Takagi
  4. Gabrielle de Wit
  5. Adam Fineberg
  6. Lachlan MacKinnon
  7. Gavin Young
  8. James R Sellers
  9. Philipp Kukura
(2015)
Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy
eLife 4:e05413.
https://doi.org/10.7554/eLife.05413
  2. Download BibTeX
  3. Download .RIS

Abstract

Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds actin in a controlled and manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an angstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head-head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps.

Article and author information

Author details

  1. Joanna Andrecka

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  2. Jaime Ortega Arroyo

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  3. Yasuharu Takagi

    Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Gabrielle de Wit

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  5. Adam Fineberg

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  6. Lachlan MacKinnon

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  7. Gavin Young

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  8. James R Sellers

    Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Philipp Kukura

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    For correspondence
    philipp.kukura@chem.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Taekjip Ha, University of Illinois, Urbana-Champaign, United States

Version history

  1. Received: November 13, 2014
  2. Accepted: March 5, 2015
  3. Accepted Manuscript published: March 6, 2015 (version 1)
  4. Version of Record published: April 9, 2015 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 3,883
    views
  • 868
    downloads
  • 67
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Joanna Andrecka
  2. Jaime Ortega Arroyo
  3. Yasuharu Takagi
  4. Gabrielle de Wit
  5. Adam Fineberg
  6. Lachlan MacKinnon
  7. Gavin Young
  8. James R Sellers
  9. Philipp Kukura
(2015)
Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy
eLife 4:e05413.
https://doi.org/10.7554/eLife.05413

Share this article

https://doi.org/10.7554/eLife.05413

Categories and tags

Further reading

    1. Structural Biology and Molecular Biophysics

    The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact

    Marco van den Noort, Panagiotis Drougkas ... Bert Poolman
    Research Article

    Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

    1. Structural Biology and Molecular Biophysics

    A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation

    Xiao-Ru Chen, Karuna Dixit ... Tatyana I Igumenova
    Research Article

    Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and βII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCβII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

    1. Structural Biology and Molecular Biophysics

    Structural insights into the GTP-driven monomerization and activation of a bacterial LRRK2 homolog using allosteric nanobodies

    Christian Galicia, Giambattista Guaitoli ... Wim Versées
    Research Article

    Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson’s disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Several structures of LRRK2 have been reported, but structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single-particle cryo-electron microscopy to solve the structure of a bacterial Roco protein (CtRoco) in its GTP-bound state, aided by two conformation-specific nanobodies: NbRoco1 and NbRoco2. This structure presents CtRoco in an active monomeric state, featuring a very large GTP-induced conformational change using the LRR-Roc linker as a hinge. Furthermore, this structure shows how NbRoco1 and NbRoco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights into the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.