1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Light-Regulated allosteric switch enables temporal and subcellular control of enzyme activity

  1. Mark Shaaya
  2. Jordan Fauser
  3. Anastasia Zhurikhina
  4. Jason E Conage-Pough
  5. Vincent Huyot
  6. Martin Brennan
  7. Cameron T Flower
  8. Jacob Matsche
  9. Shahzeb Khan
  10. Viswanathan Natarajan
  11. Jalees Rehman
  12. Pradeep Kota
  13. Forest M White
  14. Denis Tsygankov
  15. Andrei V Karginov  Is a corresponding author
  1. University of Illinois at Chicago, United States
  2. Georgia Institute of Technology and Emory University School of Medicine, United States
  3. Massachusetts Institute of Technology, United States
  4. University of North Carolina, United States
  5. Koch Institute for Integrative Cancer Research at MIT, United States
Tools and Resources
  • Cited 5
  • Views 2,476
  • Annotations
Cite this article as: eLife 2020;9:e60647 doi: 10.7554/eLife.60647

Abstract

Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.

Data availability

The raw mass spectrometry data and associated tables have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD018162. All data generated or analyzed during this study are included in the manuscript and supporting files.

The following data sets were generated

Article and author information

Author details

  1. Mark Shaaya

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Jordan Fauser

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Anastasia Zhurikhina

    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jason E Conage-Pough

    The David H Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1614-9374
  5. Vincent Huyot

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Martin Brennan

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Cameron T Flower

    The David H Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9632-9913
  8. Jacob Matsche

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Shahzeb Khan

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Viswanathan Natarajan

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Jalees Rehman

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2787-9292
  12. Pradeep Kota

    Department of Medicine, University of North Carolina, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Forest M White

    Biological Engineering, Koch Institute for Integrative Cancer Research at MIT, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1545-1651
  14. Denis Tsygankov

    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1180-3584
  15. Andrei V Karginov

    Department of Pharmacology, University of Illinois at Chicago, Chicago, United States
    For correspondence
    karginov@uic.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2370-6383

Funding

National Institutes of Health (R21CA212907)

  • Jordan Fauser

National Institutes of Health (CA238720)

  • Forest M White

Chicago Biomedical Consortium

  • Andrei V Karginov

Army Research Office (W911NF-17-1-0395)

  • Denis Tsygankov

National Institutes of Health (R21CA159179)

  • Andrei V Karginov

National Institutes of Health (R01GM118582)

  • Andrei V Karginov

National Institutes of Health (R21CA223915)

  • Jalees Rehman
  • Andrei V Karginov

National Institutes of Health (HL007829-22)

  • Mark Shaaya
  • Jordan Fauser
  • Martin Brennan

National Institutes of Health (P01 HL060678)

  • Viswanathan Natarajan
  • Jalees Rehman
  • Andrei V Karginov

National Institutes of Health (CA210180)

  • Forest M White

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

Reviewing Editor

  1. Luis F Larrondo, Pontificia Universidad Católica de Chile, Chile

Publication history

  1. Received: July 2, 2020
  2. Accepted: September 22, 2020
  3. Accepted Manuscript published: September 23, 2020 (version 1)
  4. Accepted Manuscript updated: September 24, 2020 (version 2)
  5. Version of Record published: October 21, 2020 (version 3)
  6. Version of Record updated: November 11, 2020 (version 4)

Copyright

© 2020, Shaaya 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.

Metrics

  • 2,476
    Page views
  • 457
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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)

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

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

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Shannon J McKie et al.
    Research Article

    DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase, however robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a significant increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.

    1. Biochemistry and Chemical Biology
    Theresa Hwang et al.
    Short Report

    The human proteome is replete with short linear motifs (SLiMs) of four to six residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of human ENAH, an actin regulator that is highly expressed in invasive cancers, we screened 36-residue proteome-derived peptides and discovered new interaction partners of ENAH and diverse mechanisms by which context influences binding. A pocket on the ENAH EVH1 domain that has diverged from other Ena/VASP paralogs recognizes extended SLiMs and favors motif-flanking proline residues. Many high-affinity ENAH binders that contain two proline-rich SLiMs use a noncanonical site on the EVH1 domain for binding and display a thermodynamic signature consistent with the two-motif chain engaging a single domain. We also found that photoreceptor cilium actin regulator (PCARE) uses an extended 23-residue region to obtain a higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of proteomic context on motif-mediated binding, revealing diverse mechanisms of control over EVH1 interactions and establishing that SLiMs can’t be fully understood outside of their native context.