switchSENSE technology reveals distinct activation mechanisms for PKG I

Introduction: cGMP-dependent protein kinase I (PKG I) can be activated by its non-canonical activator cAMP. However, cAMP most likely pushes PKG I in a distinct different, yet potentially physiologically relevant conformation.Results: Binding affinities of cAMP and cGMP to PKG I were determined using surface Plasmon resonance (SPR) and different conformations of PKG Iα were measured by size exclusion chromatography in the presence and absence of cyclic nucleotides, divalent metal ions and nucleotides. The novel switchSENSE technology (Dynamic Biosensors, Munich) can be utilized to measure reversible conformational changes of PKG I directly. The technology has two modes of operation: In fluorescence proximity sensing (static) mode, changes in the proximity of a fluorophore are detected, subsequently rate constants and affinities can be calculated. In dynamic response mode, the lag-values change depending on the hydrodynamic diameter of the protein attached to the DNA nanolevers. These conformational changes can be induced by the binding of cyclic nucleotides or other effectors to the protein kinase. Both, static and dynamic mode demonstrate dose dependent conformational changes in PKG I in response to cGMP and cAMP. These differences have an impact on kinase activation (activation constant for PKG Iβ with VASPtide as substrate 200 nM for cGMP and in the μM range for cAMP). Interestingly, the maximal activity of the cAMP activated PKG I is significantly lower compared to cGMP-activated protein. Our data provide mechanistic insight into the crosstalk between cGMP- and cAMP- dependent protein kinase signaling pathways.