The amino terminus of cGMP-dependent protein kinase I? increases the dynamics of the protein's cGMP-binding pockets.
ABSTRACT: The type I cGMP-dependent protein kinases play critical roles in regulating vascular tone, platelet activation and synaptic plasticity. PKG I ? and PKG I? differ in their first ~100 amino acids giving each isoform unique dimerization and autoinhibitory domains with identical cGMP-binding pockets and catalytic domains. The N-terminal leucine zipper and autoinhibitory domains have been shown to mediate isoform specific affinity for cGMP. PKG I? has a >10 fold higher affinity for cGMP than PKG I?, and PKG I? that is missing its leucine zipper has a three-fold decreased affinity for cGMP. The exact mechanism through which the N-terminus of PKG alters cGMP-affinity is unknown. In the present study, we have used deuterium exchange mass spectrometry to study how PKG I?'s N-terminus affects the conformation and dynamics of its cGMP-binding pockets. We found that the N-terminus increases the rate of deuterium exchange throughout the cGMP-binding domain. Our results suggest that the N-terminus shifts the conformational dynamics of the binding pockets, leading to an "open" conformation that has an increased affinity for cGMP.
Project description:Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG.
Project description:Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the ?C-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.
Project description:The physiological effects of cGMP are largely determined by the activities of intracellular receptors, including cGMP-dependent protein kinase (PKG) and cGMP-binding cyclic nucleotide phosphodiesterases (PDEs), and the distribution of cGMP among these receptors dictates activity of the signalling pathway. In the present study, the effects of PKG-Ialpha or PKG-Ibeta on the rate of cGMP hydrolysis by the isolated PDE5 catalytic domain were examined. PKG-Ialpha strongly inhibited cGMP hydrolysis with an IC(50) value of 217 nM, which is similar to the physiological concentration of PKG in pig coronary artery reported previously. By contrast, PKG-Ibeta, which has lower affinity for cGMP than does PKG-Ialpha, inhibited cGMP hydrolysis with an IC(50) of approx. 1 microM. Inhibition by PKG-Ialpha was more effective than that by PKG-Ibeta, consistent with their relative affinities for cGMP. Autophosphorylation of PKGs increased their cGMP-binding affinities and their inhibitory effects on PDE5 hydrolysis of cGMP. Autophosphorylation of PKG-Ibeta increased its inhibitory potency on PDE5 hydrolysis of cGMP by 10-fold compared with a 2-fold increase upon autophosphorylation of PKG-Ialpha. The results indicate that cGMP bound to allosteric cGMP-binding sites of PKG is protected from hydrolysis by PDE5 and that persistent protection of cGMP by either non-phosphorylated or autophosphorylated PKGs may be a positive-feedback control to sustain cGMP signalling.
Project description:High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG I? CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP.
Project description:cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40-83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the β1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iβ LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.
Project description:The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.
Project description:Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG I? but not PKG I?. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG I? N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform I?, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.
Project description:The two isoforms of type I cGMP-dependent protein kinase (PKGI? and PKGI?) differ in their first ?100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The dimerization domains form coiled-coil structures and serve as platforms for isoform-specific protein-protein interactions. Using the PKGI? dimerization domain as an affinity probe in a proteomic screen, we identified the actin/myosin-associated protein caldesmon (CaD) as a PKGI?-specific binding protein. PKGI? phosphorylated human CaD on serine 12 in vitro and in intact cells. Phosphorylation on serine 12 or mutation of serine 12 to glutamic acid (S12E) reduced the interaction between CaD and myosin IIA. Because CaD inhibits myosin ATPase activity and regulates cell motility, we examined the effects of PKGI? and CaD on cell migration and invasion. Inhibition of the NO/cGMP/PKG pathway reduced migration and invasion of human breast cancer cells, whereas PKG activation enhanced their motility and invasion. siRNA-mediated knockdown of endogenous CaD had pro-migratory and pro-invasive effects in human breast cancer cells. Reconstituting cells with wild-type CaD slowed migration and invasion; however, CaD containing a phospho-mimetic S12E mutation failed to reverse the pro-migratory and pro-invasive activity of CaD depletion. Our data suggest that PKGI? enhances breast cancer cell motility and invasive capacity, at least in part, by phosphorylating CaD. These findings identify a pro-migratory and pro-invasive function for PKGI? in human breast cancer cells, suggesting that PKGI? is a potential target for breast cancer treatment.
Project description:Type I cGMP-dependent protein kinases (PKGs) translocate to the nucleus to regulate gene expression in some, but not all cell types; we hypothesized that nuclear translocation of PKG may be regulated by extra-nuclear anchoring proteins. The inositol 1,4,5-triphosphate (IP(3)) receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG Ibeta, but not PKG Ialpha, and in smooth muscle cells, IRAG and PKG Ibeta are in a complex with the IP(3) receptor at endoplasmatic reticulum membranes, where the complex regulates calcium release [Schlossmann et al., Nature, 404 (2000) 197]. We found that co-expression of IRAG and PKG Ibeta in baby hamster kidney cells prevented cGMP-induced PKG Ibeta translocation to the nucleus, and decreased cGMP/PKG Ibeta transactivation of a cAMP-response element-dependent reporter gene. These effects required the PKG Ibeta/IRAG association, as demonstrated by a binding-incompetent IRAG mutant, and were specific for PKG Ibeta, as nuclear translocation and reporter gene activation by PKG Ialpha was not affected by IRAG. A phosphorylation-deficient IRAG mutant that is no longer functionally regulated by PKG phosphorylation suppressed cGMP/PKG Ibeta transcriptional activity, indicating that IRAG's effect was not explained by changes in intracellular calcium, and was not related to competition of IRAG with other PKG substrates. These results demonstrate that PKG anchoring to a specific binding protein is sufficient to dictate subcellular localization of the kinase and affect cGMP signaling in the nucleus, and may explain why nuclear translocation of PKG I does not occur in all cell types.
Project description:The cGMP-dependent protein kinase (PKG) serves as an integral component of second messenger signaling in a number of biological contexts including cell differentiation, memory, and vasodilation. PKG is homodimeric and large conformational changes accompany cGMP binding. However, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP-induced activation remain unknown. Here, we report the 2.5 Å crystal structure of a regulatory domain construct (aa 78-355) containing both cGMP binding sites of PKG I?. A distinct and segregated architecture with an extended central helix separates the two cGMP binding domains. Additionally, a previously uncharacterized helical domain (switch helix) promotes the formation of a hydrophobic interface between protomers. Mutational disruption of this interaction in full-length PKG implicates the switch helix as a critical site of dimer communication in PKG biology. These results offer new structural insight into the mechanism of allosteric PKG activation.