1. Structural Biology and Molecular Biophysics
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Deconstruction of the Ras switching cycle through saturation mutagenesis

  1. Pradeep Bandaru
  2. Neel H Shah
  3. Moitrayee Bhattacharyya
  4. John P Barton
  5. Yasushi Kondo
  6. Joshua C Cofsky
  7. Christine L Gee
  8. Arup K Chakraborty
  9. Tanja Kortemme
  10. Rama Ranganathan  Is a corresponding author
  11. John Kuriyan  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Ragon Institute of MGH, MIT and Harvard, United States
  3. University of California, San Francisco, United States
  4. University of Texas Southwestern Medical Center, United States
Research Article
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Cite this article as: eLife 2017;6:e27810 doi: 10.7554/eLife.27810

Abstract

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins.

Article and author information

Author details

  1. Pradeep Bandaru

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9354-3340

  2. Neel H Shah

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1186-0626

  3. Moitrayee Bhattacharyya

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  4. John P Barton

    Ragon Institute of MGH, MIT and Harvard, Cambridge, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1467-421X

  5. Yasushi Kondo

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  6. Joshua C Cofsky

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  7. Christine L Gee

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2632-6418

  8. Arup K Chakraborty

    Ragon Institute of MGH, MIT and Harvard, Cambridge, United States
    Competing interests
    Arup K Chakraborty, Senior editor, eLife.

  9. Tanja Kortemme

    Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  10. Rama Ranganathan

    Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    rama.ranganathan@utsouthwestern.edu
    Competing interests
    No competing interests declared.

  11. John Kuriyan

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jkuriyan@mac.com
    Competing interests
    John Kuriyan, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4414-5477

Funding

Howard Hughes Medical Institute

  • Pradeep Bandaru
  • Neel H Shah
  • Moitrayee Bhattacharyya
  • Yasushi Kondo
  • Joshua C Cofsky
  • Christine L Gee

National Institutes of Health

  • John P Barton
  • Arup K Chakraborty

Damon Runyon Cancer Research Foundation

  • Neel H Shah

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

Reviewing Editor

  1. Alfonso Valencia, Barcelona Supercomputing Center - BSC, Spain

Publication history

  1. Received: April 14, 2017
  2. Accepted: July 5, 2017
  3. Accepted Manuscript published: July 7, 2017 (version 1)
  4. Version of Record published: August 1, 2017 (version 2)

Copyright

© 2017, Bandaru et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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