Volume 16 Supplement 1

Abstracts from the 7th International Conference on cGMP Generators, Effectors and Therapeutic Implications

Open Access

Structural basis of cyclic nucleotide selectivity in cGMP dependent protein kinase II

  • James C. Campbell1,
  • Kevin Y. Li2,
  • Jeong Joo Kim3,
  • Gilbert Huang4,
  • Albert S. Reger3,
  • Shinya Matsuda5,
  • Banumathi Sankaran6,
  • Todd M. Link7,
  • Keizo Yuasa5,
  • John E. Ladbury1, 7 and
  • Choel Kim1, 3, 4Email author
BMC Pharmacology and Toxicology201516(Suppl 1):A15

DOI: 10.1186/2050-6511-16-S1-A15

Published: 2 September 2015

Background

As a central mediator of the natriuretic peptide-cGMP signalling cascade, membrane bound type II cGMP dependent protein kinase (PKG II) is a key regulator of bone growth, renin secretion, and memory formation. It represents an important drug target for treating osteoporosis, cystic fibrosis, and memory loss [15]. In spite of its crucial physiological roles and its importance as a therapeutic target, little is known about its mechanisms of cyclic nucleotide selectivity and activation due to a lack of structural information. PKG II contains an N-terminal regulatory (R)-domain that binds a C-terminal catalytic (C)-domain in the absence of cGMP. Binding of cGMP to the cyclic nucleotide binding domains (CNB-A and B) within the R-domain releases the C-domain, leading to activation. We sought to understand the cyclic nucleotide selectivity and activation mechanisms of PKG II by studying each CNB domain.

Methods and results

We screened and identified CNB domains of PKG II that are suitable for our structural studies using a high throughput Ligation Independent Cloning method. Our affinity measurements of the resulting CNB domains showed that CNB-B binds cGMP with a higher affinity, providing almost 500-fold selectivity, while CNB-A only offers 10-fold selectivity [6]. To understand the structural basis of each domain's cGMP selectivity, we solved crystal structures of CNB-A and -B in the presence of cyclic nucleotides. The structures revealed that only CNB-B shows an ordered C-helix that shields the cGMP pocket and specifically interacts with the guanine moiety through several hydrogen bond and VWD contacts. In contrast, CNB-A displays an open pocket without a C-terminal helix, resulting in fewer interactions with cGMP. Our mutation analysis demonstrated that the polar contacts at the C-helix of CNB-B are crucial for high cGMP selectivity and kinase activation.

Conclusion

Our structural comparison with cGMP selective PKG I CNB-B domain shows that it lacks cGMP specific hydrogen bonding contacts at the C-helix, which suggests a distinct cGMP selectivity mechanism for PKG II's CNB-B (Figure 1). Cyclic nucleotide compartmentalization is crucial for signalling specificity and exists in both cGMP and cAMP pathways [710]. Due to higher cAMP concentrations at the cell membrane compared to the cytosol, the higher cGMP selectivity seen in CNB-B of PKG II might be important in preventing activation of PKG II by cAMP, and this might minimize undesired cross-activation of both cyclic nucleotide signalling pathways.
Figure 1

Isotype specific cGMP selectivity mechanisms of type I and II PKGs

Authors’ Affiliations

(1)
Structural and Computational Biology and Molecular Biophysics Program, Baylor College of Medicine
(2)
Department of Biochemistry & Cell Biology, Rice University
(3)
Department of Pharmacology, Baylor College of Medicine
(4)
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine
(5)
Department of Biological Science and Technology, The University of Tokushima Graduate School
(6)
Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory
(7)
Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center

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Copyright

© Campbell et al. 2015

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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