Structural Basis for Cyclic-Nucleotide Selectivity and cGMP-Selective Activation of PKG I

Abstract

cGMP and cAMP-dependent protein kinases (PKG and PKA) are closely related homologs, and the cyclic nucleotide specificity of each kinase is crucial for keeping the two signaling pathways segregated, but the molecular mechanism of cyclic nucleotide selectivity is unknown. Here we report that the PKG Iβ C-terminal cyclic nucleotide binding domain (CNB-B) is highly selective for cGMP binding, and have solved crystal structures of CNB-B with and without bound cGMP. These structures, combined with a comprehensive mutagenic analysis, allowed us to identify Leu296 and Arg297 as key residues which mediate cGMP selectivity. In addition, by comparing the cGMP bound and unbound structures, we observed large conformational changes in the C-terminal helices in response to cGMP binding, which were stabilized by recruitment of Tyr351 as a “capping residue” for cGMP. The observed rearrangements of the C-terminal helices provide a mechanical insight into release of the catalytic domain and kinase activation.

Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG)

“…Indeed, the C-terminal cyclic nucleotide-binding domain (CNB-B) of PKG I␤ was previously found to exhibit greater binding affinity for cGMP, which has been attributed to specific interactions between the base-binding region (BBR) 5 of CNB-B and the cGMP base moiety, as observed in previously solved crystallographic x-ray structures ( Fig. 1e) (17,18). However, the intracellular concentration of cAMP is typically significantly greater than that of cGMP (19 -23), thus 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.…”

“…Such partial agonism of cAMP is not fully explained by previously solved structures of PKG I␤, as they reveal very similar structural rearrangements of both CNB domains in response to either cAMP or cGMP binding (Fig. 1, b-d) (2,17,18) that are analogous to structural rearrangements observed for other CNB domains (8, 12, 24 -29). Hence, we hypothesize that the partial agonist response of PKG I␤ to cAMP may reflect differences in the dynamics of cAMP versus cGMP-bound PKG I␤.…”

“…1a), and it has been previously identified as a critical control unit for auto-inhibition and cGMP selectivity in PKG I␤ (17,18). In addition, we examined the CNB-B domain because although the CNB-A domain contributes to the cyclic nucleotide modulation of PKG I function (17,(35)(36)(37), the CNB-A domain undergoes only minor structural changes upon cyclic nucleotide binding (2,17). We found that the partial agonism of cAMP cannot be explained by a simple reversal of the two-state inactive/active conformational equilibrium that rationalizes cGMP activation (2,17).…”

“…In addition, we examined the CNB-B domain because although the CNB-A domain contributes to the cyclic nucleotide modulation of PKG I function (17,(35)(36)(37), the CNB-A domain undergoes only minor structural changes upon cyclic nucleotide binding (2,17). We found that the partial agonism of cAMP cannot be explained by a simple reversal of the two-state inactive/active conformational equilibrium that rationalizes cGMP activation (2,17). Rather, our data show that Residue numbers for the regulatory region domain boundaries are indicated, and the monomer fragment examined in the current study (referred to herein as PKG I␤(219 -369)) is highlighted in gray.…”

“…Rather, our data show that Residue numbers for the regulatory region domain boundaries are indicated, and the monomer fragment examined in the current study (referred to herein as PKG I␤(219 -369)) is highlighted in gray. b-d, ribbon structure overlays of the previously solved apo (red ribbon), cAMP-bound (blue ribbon), and cGMP-bound (green ribbon) structures of PKG I␤(219 -369) (17,18). Bound cAMP and cGMP are shown as blue and green sticks, respectively, and the following key structural elements are indicated: the N-terminal ␣-helix bundle (N3A), the ␤-barrel (␤-core); the BBR element involved in cGMP binding; and the C-terminal ␣B-helix (␣B) and switch helix region.…”