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

Rajesh Sharma, Jeong Joo Kim, Liying Qin, Philipp Henning, Madoka Akimoto, Bryan VanSchouwen, Gundeep Kaur, Banumathi Sankaran, Kevin R. MacKenzie, Giuseppe Melacini, View ORCID ProfileDarren E. Casteel, Friedrich W. Herberg, View ORCID ProfileChoel Kim
doi: https://doi.org/10.1101/2022.04.28.489861
Rajesh Sharma
aDepartment of Pharmacology and Chemical Biology and Center for Drug Discovery, Baylor College of Medicine, Houston, TX, USA
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Jeong Joo Kim
aDepartment of Pharmacology and Chemical Biology and Center for Drug Discovery, Baylor College of Medicine, Houston, TX, USA
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Liying Qin
aDepartment of Pharmacology and Chemical Biology and Center for Drug Discovery, Baylor College of Medicine, Houston, TX, USA
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Philipp Henning
bDepartment of Biochemistry, University of Kassel, Kassel, Germany
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Madoka Akimoto
cDepartment of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada
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Bryan VanSchouwen
cDepartment of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada
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Gundeep Kaur
aDepartment of Pharmacology and Chemical Biology and Center for Drug Discovery, Baylor College of Medicine, Houston, TX, USA
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Banumathi Sankaran
dBerkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
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Kevin R. MacKenzie
aDepartment of Pharmacology and Chemical Biology and Center for Drug Discovery, Baylor College of Medicine, Houston, TX, USA
eDepartment of Pathology and Immunology and Center for Drug Discovery, Baylor College of Medicine, Houston, TX, USA
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  • For correspondence: ckim@bcm.edu km5@bcm.edu
Giuseppe Melacini
cDepartment of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada
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Darren E. Casteel
fDepartment of Medicine, University of California, San Diego, La Jolla, CA 92093 USA
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  • ORCID record for Darren E. Casteel
Friedrich W. Herberg
bDepartment of Biochemistry, University of Kassel, Kassel, Germany
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Choel Kim
aDepartment of Pharmacology and Chemical Biology and Center for Drug Discovery, Baylor College of Medicine, Houston, TX, USA
gVerna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
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  • ORCID record for Choel Kim
  • For correspondence: ckim@bcm.edu km5@bcm.edu
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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. Campaigns targeting nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis suggest that understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs. 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 still 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 activation of PKGs.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted April 28, 2022.
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An auto-inhibited state of protein kinase G and implications for selective activation
Rajesh Sharma, Jeong Joo Kim, Liying Qin, Philipp Henning, Madoka Akimoto, Bryan VanSchouwen, Gundeep Kaur, Banumathi Sankaran, Kevin R. MacKenzie, Giuseppe Melacini, Darren E. Casteel, Friedrich W. Herberg, Choel Kim
bioRxiv 2022.04.28.489861; doi: https://doi.org/10.1101/2022.04.28.489861
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An auto-inhibited state of protein kinase G and implications for selective activation
Rajesh Sharma, Jeong Joo Kim, Liying Qin, Philipp Henning, Madoka Akimoto, Bryan VanSchouwen, Gundeep Kaur, Banumathi Sankaran, Kevin R. MacKenzie, Giuseppe Melacini, Darren E. Casteel, Friedrich W. Herberg, Choel Kim
bioRxiv 2022.04.28.489861; doi: https://doi.org/10.1101/2022.04.28.489861

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