Abstract
Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP versus cAMP is critical. However, the mechanism of cGMP-versus-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic nucleotide-binding domain B of PKG binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-versus-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP-, and cAMP-bound forms of the PKG cyclic nucleotide-binding domain B. We show that although cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state.
Highlights
Protein kinase G (PKG) is selectively activated by cGMP, but the mechanism of cGMP-versus-cAMP selectivity is not fully understood
We show that cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state
Chemical shifts from the apo state revealed that binding of both cAMP and cGMP results in major widespread structural changes within the cyclic nucleotide-binding domain (CNB-B) domain, including part of the switch helix region (Fig. 2c, black and green points), in agreement with the available x-ray structures [17, 18]
Summary
Protein kinase G (PKG) is selectively activated by cGMP, but the mechanism of cGMP-versus-cAMP selectivity is not fully understood. The intracellular concentration of cAMP is typically significantly greater than that of cGMP (19 –23), potentially allowing cAMP to bind to PKG despite its lower binding affinity and suggesting that other means of cyclic nucleotide selectivity must be present in PKG Another key difference in the response of PKG I to cAMP versus cGMP is that in contrast to cGMP, cAMP is only a partial agonist of PKG I, as shown by Cook photometric kinase assays of PKG I activity (Fig. 1f). In the context of the two-state conformational selection mechanism, the partial agonism of cAMP could arise from a reduced inactive-to-active shift of the dynamic conformational equilibrium underlying PKG activation (30 –33) Such conformational dynamics have been shown to play a role in the varying responses of other CNB domains to cyclic nucleotides [13, 34]. By populating this third conformation, and to a lesser degree the inactive conformation, cAMP reduces the overall level of PKG activity compared to cGMP, thereby providing an explanation for the observed partial agonism of cAMP
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