The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an assay, we observed that oxidation with hydrogen peroxide (HO) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by HO In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα-specific substrate, the large conductance potassium channel (K 1.1). Application of WT PKG Iα activated by either cGMP or HO increased the open probabilities of the channel. Neither cGMP nor HO activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased K 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered K 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

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.

The type I cGMP-dependent protein kinases serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMPdependent protein kinase (PKA). Here, we examined PKG Ia activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 ( 53). We found that the construct exists as a monomer as assessed by whole-protein mass spectrometry, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that 53 has a similar shape to the heterodimeric RIa:C complex of PKA. Moreover, we found that the 53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length as assessed by surface plasmon resonance spectroscopy (SPR). However we found that the 53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Ia activation is distinct from PKA, and its cooperativity is driven by in trans interactions between protomers.