A general strategy to construct small molecule biosensors in eukaryotes

  1. Justin Feng
  2. Benjamin W Jester
  3. Christine E Tinberg
  4. Daniel J Mandell  Is a corresponding author
  5. Mauricio S Antunes
  6. Raj Chari
  7. Kevin J Morey
  8. Xavier Rios
  9. June I Medford
  10. George M Church
  11. Stanley Fields
  12. David Baker
  1. Harvard Medical School, United States
  2. University of Washington, United States
  3. Colorado State University, United States
  4. Howard Hughes Medical Institute, University of Washington, United States

Abstract

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

Article and author information

Author details

  1. Justin Feng

    Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, United States
    Competing interests
    Justin Feng, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work.
  2. Benjamin W Jester

    Department of Genome Sciences, University of Washington, Seattle, United States
    Competing interests
    Benjamin W Jester, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work.
  3. Christine E Tinberg

    Department of Biochemistry, University of Washington, Seattle, United States
    Competing interests
    Christine E Tinberg, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work.
  4. Daniel J Mandell

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    djmandell@gmail.com
    Competing interests
    Daniel J Mandell, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work. Harvard University has filed a provisional patent.
  5. Mauricio S Antunes

    Department of Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  6. Raj Chari

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    Raj Chari, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work.
  7. Kevin J Morey

    Department of Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  8. Xavier Rios

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  9. June I Medford

    Department of Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  10. George M Church

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    George M Church, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work.
  11. Stanley Fields

    Department of Genome Sciences, University of Washington, Seattle, United States
    Competing interests
    Stanley Fields, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work.
  12. David Baker

    Howard Hughes Medical Institute, University of Washington, Seattle, United States
    Competing interests
    David Baker, has filed a provisional application (patent application number 62220628) with the US Patent and Trademark Office on this work.

Reviewing Editor

  1. Jeffery W Kelly, Scripps Research Institute, United States

Publication history

  1. Received: August 6, 2015
  2. Accepted: December 17, 2015
  3. Accepted Manuscript published: December 29, 2015 (version 1)
  4. Accepted Manuscript updated: December 30, 2015 (version 2)
  5. Version of Record published: January 26, 2016 (version 3)

Copyright

© 2015, Feng 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

  • 12,451
    Page views
  • 3,036
    Downloads
  • 106
    Citations

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

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. Justin Feng
  2. Benjamin W Jester
  3. Christine E Tinberg
  4. Daniel J Mandell
  5. Mauricio S Antunes
  6. Raj Chari
  7. Kevin J Morey
  8. Xavier Rios
  9. June I Medford
  10. George M Church
  11. Stanley Fields
  12. David Baker
(2015)
A general strategy to construct small molecule biosensors in eukaryotes
eLife 4:e10606.
https://doi.org/10.7554/eLife.10606

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Rajesh Sharma et al.
    Research Article

    Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here we present a crystal structure of PKG Iβ in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yitong Li et al.
    Research Article

    Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryo-EM structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multi-partite contacts at structured cores to activate the methylesterase. B56-interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56-interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.