Co-Crystal Structures of PKG Iβ (92–227) with cGMP and cAMP Reveal the Molecular Details of Cyclic-Nucleotide Binding

Abstract

BackgroundCyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively.Methodology/Principal FindingsWe have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A) of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP.Conclusions/SignificanceOur findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.

Mechanism of cAMP Partial Agonism in Protein Kinase G (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␤.…”

“…By binding to PKG, cGMP regulates intracellular signaling pathways that control a wide range of intracellular processes, such as cell differentiation, platelet activation, memory formation, and vasodilation (1)(2)(3)(4)(5). The PKG signaling pathways are often distinct from those regulated by cAMP-dependent proteins such as protein kinase A (PKA) (6 -12).…”

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

“…The cAMP-bound structure of the HCN carboxyl-terminal region has been solved for several HCN channels (10)(11)(12)(13)(14) and is similar to the agonist-bound structures of many other CNBDcontaining proteins (15)(16)(17)(18)(19). However, the agonist-free structure of this protein is still controversial.…”

“…Each of the subunits contains two domains: the cyclic nucleotide-binding domain (CNBD) and the C-linker domain. The CNBD exhibits strong structural similarity to the CNBDs of other cyclic nucleotide-binding proteins, including cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac, and the Escherichia coli catabolite gene activator protein (CAP) (15)(16)(17)(18)(19). The CNBD consists of an eight-stranded antiparallel β-roll, followed by two α-helices (B-helix and C-helix).…”

A Mechanism for the Auto-inhibition of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Opening and Its Relief by cAMP

“…ϭ 0.5 Å), with the exception of partial uncoiling of the FЈ-helix and of the C-terminal half of the C-helix, which was unresolved (25). However, it has been suggested that the similarity between the apo-and holo-HCN2 IR tetramer structures might at least in part be due to crystal packing and a co-crystallized bromide ion mimicking the cAMP phosphate (25), as also observed for other apo-CBDs (26,27). Furthermore, size exclusion chromatography and analytical ultracentrifugation indicate that in solution the IR of HCN2 and HCN4 is primarily monomeric in the apo-form, with minimal or no tetrameric IR detected (11,21).…”