Towards a Mechanism of Oxidation-Dependent Regulation of cGMP-Dependent Protein Kinase Iα

Abstract

cGMP-dependent protein kinase Iα (PKG) is a key regulator of blood pressure and a potential target of antihypertensive drugs. PKG activity is tightly controlled by cGMP, a secondary messenger in the nitric oxide and natriuretic peptide signalling pathways. However, the cGMP-dependent activation of PKG is modulated by oxidative stress. At low cGMP levels, oxidation causes constitutive activation of PKG, while at higher cGMP levels oxidation reduces PKG kinase activity. Understanding the oxidation-dependent modulation of PKG is critical to rationalize the mechanism of action for a new class of antihypertensive drugs. However, the molecular mechanism underlying the redox sensitivity of PKG is currently not fully understood. Here, using Nuclear Magnetic Resonance spectroscopy (NMR), we show that oxidation results not only in a reversible disulfide bridge (C118-C196) in cyclic nucleotide-binding domain A (CNB-A) of PKG, but also in a rewiring of the allosteric networks that connect this disulfide to the phosphate binding cassette (PBC), a critical hotspot conserved among CNBs that controls both inhibitory interactions with the catalytic domain in the absence of cGMP and dis-inhibitory inter-protomer interactions in the presence of cGMP. Moreover, we show how oxidation impacts the dynamics of both cGMP-bound CNB-A, particularly within the PBC, potentially destabilizing the active state of PKG, and inhibitor-bound CNB-A, destabilizing the inactive state of PKG. The proposed model accounts for both the attenuated cGMP-dependent activation observed for the oxidized kinase and the oxidation-induced constitutive activation of PKG. These results provide a framework to understand the mechanism of action of clinical anti-hypertensive drug leads that target PKG and illustrate how redox-dependent allosteric networks modulate kinase activity.