cGMP-dependent protein kinase (PKG) is the central enzyme of NO-cGMP signaling pathway that regulates platelet aggregation, smooth muscle tone, phototransduction, and leukocyte migration [1,2]. Although PKG has been heavily targeted for treating diseases such as, erectile dysfunction and cardiovascular and pulmonary diseases, developing specific activators and inhibitors has been difficult because there is no structural information available[1,2]. For the successful NO-cGMP mediated signaling responses to occur, PKG has to be localized to the specific site in the cell. Localization of PKG is an essential feature of the NO-cGMP signaling and mediated by G Kinase Anchoring Proteins (GKAPs)[3,4,5,6,7,8]. In particular, the variable leucine zipper domain at the extreme N-terminus targets PKG to specific subcellular sites via its interaction with G-kinase anchoring proteins in an isotype specific manner [7,9]. Despite mounting evidence that GKAP target PKGs via its association with the zipper domain, the molecular details of this important protein- protein interaction remain unknown.
The specific aims of this proposal are to understand the isotype specific targeting mechanism of PKGs using peptide arrays in combination with X-ray crystallography. Adding another layer of complexity, there are three types of PKG (I1, I2, and II) that each have different cGMP dependence in activation, unique sets of substrates, and tissue specific expression [2,10,11]. My primary focus is the type I isozymes (1 and 2) that have been implicated in erectile dysfunction and many cardiovascular diseases. Although they represent functionally non- redundant proteins with unique physiological roles, their functional roles and specific subcellular localization are poorly understood. In order to elucidate isozyme specific functions and localization, I plan to solve crystal structures of the PKG I1 and 2 zipper domains and compare molecular features of GKAP docking surfaces that are unique to each isoform. In parallel, I plan to engineer peptides that can selectively disrupt interactions between both isozymes of PKG I and their individual binding partners. Lastly, I will incorporate high affinity peptides into the co-crystallization trials in order to understand molecular details of the PKG/GKAP interaction. My plan is to form stable protein/peptide complexes using isozyme specific peptides and pursue high-resolution crystal structures of PKG/GAKP complexes. Solution of the zipper domain structures, and development of small peptides that specifically disrupt isozyme specific targeting, will pave the way to elucidation of the specific functions of PKGs and eventually lead to the development of therapeutic agents.
cGMP-dependent protein kinase (PKG) is the central enzyme of NO-cGMP signaling pathway that regulates platelet aggregation, smooth muscle tone, phototransduction, and leukocyte migration. Although PKG has been heavily targeted for treating diseases such as, erectile dysfunction and cardiovascular and pulmonary diseases, developing specific activators and inhibitors has been difficult because there is no structural information available. My ultimate goal is to rationally target the kinase by obtaining high-resolution crystal structures of PKG and its isozymes and develop pharmacological agents that can modulate the activity of the kinase to treat diseases related to NO-cGMP signaling dysfunction.
|Gerlits, Oksana; Campbell, James C; Blakeley, Matthew P et al. (2018) Neutron Crystallography Detects Differences in Protein Dynamics: Structure of the PKG II Cyclic Nucleotide Binding Domain in Complex with an Activator. Biochemistry 57:1833-1837|
|Qin, Liying; Sankaran, Banumathi; Aminzai, Sahar et al. (2018) Structural basis for selective inhibition of human PKG I? by the balanol-like compound N46. J Biol Chem 293:10985-10992|
|Campbell, James C; VanSchouwen, Bryan; Lorenz, Robin et al. (2017) Crystal structure of cGMP-dependent protein kinase I? cyclic nucleotide-binding-B domain : Rp-cGMPS complex reveals an apo-like, inactive conformation. FEBS Lett 591:221-230|
|Campbell, James C; Henning, Philipp; Franz, Eugen et al. (2017) Structural Basis of Analog Specificity in PKG I and II. ACS Chem Biol 12:2388-2398|
|Lorenz, Robin; Moon, Eui-Whan; Kim, Jeong Joo et al. (2017) Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity. Biochem J 474:2389-2403|
|He, Daniel; Lorenz, Robin; Kim, Choel et al. (2017) Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains. ACS Chem Biol 12:3057-3066|
|Campbell, James C; Kim, Jeong Joo; Li, Kevin Y et al. (2016) Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II. J Biol Chem 291:5623-33|
|Kim, Jeong Joo; Lorenz, Robin; Arold, Stefan T et al. (2016) Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation. Structure 24:710-720|
|Kim, Jeong Joo; Flueck, Christian; Franz, Eugen et al. (2015) Crystal structures of the carboxyl cGMP binding domain of the Plasmodium falciparum cGMP-dependent protein kinase reveal a novel capping triad crucial for merozoite egress. PLoS Pathog 11:e1004639|
|VanSchouwen, Bryan; Selvaratnam, Rajeevan; Giri, Rajanish et al. (2015) Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG). J Biol Chem 290:28631-41|
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