The role of the low affinity cyclic nucleotide-binding site B in the activation of cGMP-dependent protein kinase Iα

Cyclic guanosine 3’,5’-monophosphate (cGMP)-dependent protein kinase (PKG) activates a signaling pathway that leads to vascular smooth muscle cell relaxation, a process that reduces blood pressure. This enzyme consists of a dimerization domain, autoinhibitory domain, regulatory domain, and catalytic domain1. PKG is activated by cGMP binding to two binding sites of the regulatory domain. In order to study how each of these two binding sites, A and B, contributes to PKG activation, a mutant that knocked out cGMP binding to the B site, PKG Iα E292A, was expressed in Sf9 cells and purified to apparent homogeneity. Despite the presence of this mutation, the affinity for cGMP determined by surface plasmon resonance (SPR) was unchanged. The mutant still displayed cGMP dependent activation. In addition to these cGMP-binding sites, the regulatory domain contains a switch helix motif that provides a place for crosstalk between the PKG protomers2. It is not well known how this motif affects cyclic nucleotide binding. In order to determine this, cGMP binding was compared between a regulatory domain construct containing the switch helix and one without the switch helix, expressed and purified in E. coli. The presence of the switch helix hindered cyclic nucleotide binding to the regulatory domain