1. Structural Biology and Molecular Biophysics

Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas

  1. Alison B Hickman
  2. Shweta Kailasan
  3. Pavol Genzor
  4. Astrid D Haase
  5. Fred Dyda  Is a corresponding author
  1. National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
https://doi.org/10.7554/eLife.50004
Share this article
Cite this article
  1. Alison B Hickman
  2. Shweta Kailasan
  3. Pavol Genzor
  4. Astrid D Haase
  5. Fred Dyda
(2020)
Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas
eLife 9:e50004.
https://doi.org/10.7554/eLife.50004
  2. Download BibTeX
  3. Download .RIS

Abstract

Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.

Data availability

Diffraction data for the casposase-DNA complex have been deposited under PBD ID 6OPM. The GSE number for the NGS data is GSE139037.

The following data sets were generated

Article and author information

Author details

  1. Alison B Hickman

    Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7666-0249

  2. Shweta Kailasan

    Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0876-6812

  3. Pavol Genzor

    Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Astrid D Haase

    Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Fred Dyda

    Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States
    For correspondence
    Fred.Dyda@nih.gov
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1689-9041

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (Intramural Program)

  • Alison B Hickman
  • Shweta Kailasan
  • Pavol Genzor
  • Astrid D Haase
  • Fred Dyda

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Blake Wiedenheft, Montana State University, United States

Version history

  1. Received: July 8, 2019
  2. Accepted: January 8, 2020
  3. Accepted Manuscript published: January 8, 2020 (version 1)
  4. Version of Record published: January 23, 2020 (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

  • 2,925
    views
  • 489
    downloads
  • 16
    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. Alison B Hickman
  2. Shweta Kailasan
  3. Pavol Genzor
  4. Astrid D Haase
  5. Fred Dyda
(2020)
Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas
eLife 9:e50004.
https://doi.org/10.7554/eLife.50004

Share this article

https://doi.org/10.7554/eLife.50004

Categories and tags

Further reading

    1. Structural Biology and Molecular Biophysics

    Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation

    Hitendra Negi, Aravind Ravichandran ... Ranabir Das
    Research Article

    The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Special Issue: Allosteric regulation of kinase activity

    Amy H Andreotti, Volker Dötsch
    Editorial

    The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

    1. Developmental Biology
    2. Structural Biology and Molecular Biophysics

    Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling

    Samuel C Griffiths, Jia Tan ... Hsin-Yi Henry Ho
    Research Article Updated

    The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.