An auto-inhibited state of protein kinase G and implications for selective activation

  1. Rajesh Sharma
  2. Jeong Joo Kim
  3. Liying Qin
  4. Philipp Henning
  5. Madoka Akimoto
  6. Bryan VanSchouwen
  7. Gundeep Kaur
  8. Banumathi Sankaran
  9. Kevin R MacKenzie  Is a corresponding author
  10. Giuseppe Melacini
  11. Darren E Casteel
  12. Fritz W Herberg
  13. Choel W Kim  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. University of Kassel, Germany
  3. McMaster University, Canada
  4. Lawrence Berkeley National Laboratory, United States
  5. University of California, San Diego, United States

Abstract

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.

Data availability

Diffraction data have been deposited in PDB under the accession codes 7LV3 and 7MBJ.

The following data sets were generated

Article and author information

Author details

  1. Rajesh Sharma

    Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Jeong Joo Kim

    Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Liying Qin

    Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Philipp Henning

    Department of Biochemistry, University of Kassel, kassel, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Madoka Akimoto

    Department of Chemistry and Chemical Biology, McMaster University, Ontario, Canada
    Competing interests
    The authors declare that no competing interests exist.
  6. Bryan VanSchouwen

    Department of Chemistry and Chemical Biology, McMaster University, Ontario, Canada
    Competing interests
    The authors declare that no competing interests exist.
  7. Gundeep Kaur

    Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Banumathi Sankaran

    Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Kevin R MacKenzie

    Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, United States
    For correspondence
    kevin.mackenzie@bcm.edu
    Competing interests
    The authors declare that no competing interests exist.
  10. Giuseppe Melacini

    Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Canada
    Competing interests
    The authors declare that no competing interests exist.
  11. Darren E Casteel

    Department of Medicine, University of California, San Diego, San Diego, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7673-6597
  12. Fritz W Herberg

    Department of Biochemistry, University of Kassel, kassel, Germany
    Competing interests
    The authors declare that no competing interests exist.
  13. Choel W Kim

    Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, United States
    For correspondence
    ckim@bcm.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3152-0020

Funding

National Institute of General Medical Sciences (R01 GM090161)

  • Choel W Kim

Deutsche Forschungsgemeinschaft (He1818/10-1)

  • Fritz W Herberg

Federal Ministry of Education and Research, Germany (TargetRD,FKZ: 16GW0270)

  • Fritz W Herberg

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

Reviewing Editor

  1. Tony Hunter, Salk Institute for Biological Studies, United States

Publication history

  1. Received: April 15, 2022
  2. Accepted: August 4, 2022
  3. Accepted Manuscript published: August 5, 2022 (version 1)

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.

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  1. Rajesh Sharma
  2. Jeong Joo Kim
  3. Liying Qin
  4. Philipp Henning
  5. Madoka Akimoto
  6. Bryan VanSchouwen
  7. Gundeep Kaur
  8. Banumathi Sankaran
  9. Kevin R MacKenzie
  10. Giuseppe Melacini
  11. Darren E Casteel
  12. Fritz W Herberg
  13. Choel W Kim
(2022)
An auto-inhibited state of protein kinase G and implications for selective activation
eLife 11:e79530.
https://doi.org/10.7554/eLife.79530

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