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

Authors

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

Publication Date

8-5-2022

Journal

ELife

DOI

10.7554/eLife.79530

PMID

35929723

PMCID

PMC9417419

PubMedCentral® Posted Date

8-5-2022

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Animals, Cyclic GMP, Cyclic GMP-Dependent Protein Kinase Type I, Mammals, Nitric Oxide, Phosphorylation, Protein Isoforms, Human

Abstract

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (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 (AI) 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 AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI 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 CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype 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.

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