Crystal Structure of cGMP-Dependent Protein Kinase Reveals Novel Site of Interchain Communication

Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA.
Structure (Impact Factor: 5.62). 09/2011; 19(9):1317-27. DOI: 10.1016/j.str.2011.06.012
Source: PubMed

ABSTRACT

The cGMP-dependent protein kinase (PKG) serves as an integral component of second messenger signaling in a number of biological contexts including cell differentiation, memory, and vasodilation. PKG is homodimeric and large conformational changes accompany cGMP binding. However, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP-induced activation remain unknown. Here, we report the 2.5 Å crystal structure of a regulatory domain construct (aa 78-355) containing both cGMP binding sites of PKG Iα. A distinct and segregated architecture with an extended central helix separates the two cGMP binding domains. Additionally, a previously uncharacterized helical domain (switch helix) promotes the formation of a hydrophobic interface between protomers. Mutational disruption of this interaction in full-length PKG implicates the switch helix as a critical site of dimer communication in PKG biology. These results offer new structural insight into the mechanism of allosteric PKG activation.

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    • "However, elucidating the regulatory mechanism of the PKG holoenzymes is central to guiding pharmacological discoveries relevant to the prevention and treatment of vascular diseases. Recently, we solved the crystal structure of the intact regulatory domain of PKG I (Osborne et al., 2011). This structure exposed a unique helical domain that stabilizes an unexpected dimer interface between monomers in the asymmetric unit. "
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    ABSTRACT: PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix was found to bind to and activate PKG Iα in a cGMP-independent manner. The most potent S-tide derivative (S1.5) increased the open probability (NPo) of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization (RP) procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels, S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease.
    Full-text · Article · Dec 2015 · Chemistry & Biology
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    • "interventions that prevent either of their formation limiting PKG signaling responses to oxidants . Studies with metal ion - induced oxidation identified the intraprotein disulfides , whilst those with H 2 O 2 ( Landgraf et al . , 1991 ; Osborne et al . , 2011 ) , nitrosocysteine ( Burgoyne and Eaton , 2009 ) , or H 2 S ( Landgraf et al . , 1991 ; Osborne et al . , 2011 ) identified the interprotein disulfide . It is possible that each of these oxidants simultaneously induced all of the several disulfides that can form , but they were not reported in some studies because they could not be determined or were not specifically assessed ."
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    ABSTRACT: Elevated levels of oxidants in biological systems have been historically referred to as "oxidative stress," a choice of words that perhaps conveys an imbalanced view of reactive oxygen species in cells and tissues. The term stress suggests a harmful role, whereas a contemporary view is that oxidants are also crucial for the maintenance of homeostasis or adaptive signaling that can actually limit injury. This regulatory role for oxidants is achieved in part by them inducing oxidative post-translational modifications of proteins which may alter their function or interactions. Such mechanisms allow changes in cell oxidant levels to be coupled to regulated alterations in enzymatic function (i.e., signal transduction), which enables "redox signaling." In this review we focus on the role of cGMP-dependent protein kinase (PKG) Ia disulfide dimerisation, an oxidative modification that is induced by oxidants that directly activates the enzyme, discussing how this impacts on the cardiovascular system. Additionally, how this oxidative activation of PKG may coordinate with or differ from classical activation of this kinase by cGMP is also considered.
    Full-text · Article · Jul 2015 · Frontiers in Pharmacology
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    • "The cAMP-dependent protein kinases (PKAs) and cGMP-dependent protein kinases (PKGs) are part of the same sub-family of kinase domains [36] and have similar domain components, and quaternary structure [66]. PKG is composed of a single protein with cGMP binding and protein-kinase activity. "
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    ABSTRACT: Background The current knowledge of eukaryote signalling originates from phenotypically diverse organisms. There is a pressing need to identify conserved signalling components among eukaryotes, which will lead to the transfer of knowledge across kingdoms. Two useful properties of a eukaryote model for signalling are (1) reduced signalling complexity, and (2) conservation of signalling components. The alga Ostreococcus tauri is described as the smallest free-living eukaryote. With less than 8,000 genes, it represents a highly constrained genomic palette. Results Our survey revealed 133 protein kinases and 34 protein phosphatases (1.7% and 0.4% of the proteome). We conducted phosphoproteomic experiments and constructed domain structures and phylogenies for the catalytic protein-kinases. For each of the major kinases families we review the completeness and divergence of O. tauri representatives in comparison to the well-studied kinomes of the laboratory models Arabidopsis thaliana and Saccharomyces cerevisiae, and of Homo sapiens. Many kinase clades in O. tauri were reduced to a single member, in preference to the loss of family diversity, whereas TKL and ABC1 clades were expanded. We also identified kinases that have been lost in A. thaliana but retained in O. tauri. For three, contrasting eukaryotic pathways – TOR, MAPK, and the circadian clock – we established the subset of conserved components and demonstrate conserved sites of substrate phosphorylation and kinase motifs. Conclusions We conclude that O. tauri satisfies our two central requirements. Several of its kinases are more closely related to H. sapiens orthologs than S. cerevisiae is to H. sapiens. The greatly reduced kinome of O. tauri is therefore a suitable model for signalling in free-living eukaryotes. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-640) contains supplementary material, which is available to authorized users.
    Full-text · Article · Aug 2014 · BMC Genomics
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