cGMP-binding Domains Research Articles

Mutations within the cGMP-binding domain of CNGA1 causing autosomal recessive retinitis pigmentosa in human and animal model

Retinitis pigmentosa is a group of progressive inherited retinal dystrophies that may present clinically as part of a syndromic entity or as an isolated (nonsyndromic) manifestation. In an Indian family suffering from retinitis pigmentosa, we identified a missense variation in CNGA1 affecting the cyclic nucleotide binding domain (CNBD) and characterized a mouse model developed with mutated CNBD. A gene panel analysis comprising 105 known RP genes was used to analyze a family with autosomal-recessive retinitis pigmentosa (arRP) and revealed that CNGA1 was affected. From sperm samples of ENU mutagenesis derived F1 mice, we re-derived a mutant with a Cnga1 mutation. Homozygous mutant mice, developing retinal degeneration, were examined for morphological and functional consequences of the mutation. In the family, we identified a rare CNGA1 variant (NM_001379270.1) c.1525 G > A; (p.Gly509Arg), which co-segregated among the affected family members. Homozygous Cnga1 mice harboring a (ENSMUST00000087213.12) c.1526 A > G (p.Tyr509Cys) mutation showed progressive degeneration in the retinal photoreceptors from 8 weeks on. This study supports a role for CNGA1 as a disease gene for arRP and provides new insights on the pathobiology of cGMP-binding domain mutations in CNGA1-RP.

Divergent allostery reveals critical differences between structurally homologous regulatory domains of Plasmodium falciparum and human protein kinase G

Malaria is a life-threatening infectious disease primarily caused by the Plasmodium falciparum parasite. The increasing resistance to current antimalarial drugs and their side effects has led to an urgent need for novel malaria drug targets, such as the P. falciparum cGMP-dependent protein kinase (pfPKG). However, PKG plays an essential regulatory role also in the human host. Human cGMP-dependent protein kinase (hPKG) and pfPKG are controlled by structurally homologous cGMP-binding domains (CBDs). Here, we show that despite the structural similarities between the essential CBDs in pfPKG and hPKG, their respective allosteric networks differ significantly. Through comparative analyses of chemical shift covariance analyses, molecular dynamics simulations, and backbone internal dynamics measurements, we found that conserved allosteric elements within the essential CBDs are wired differently in pfPKG and hPKG to implement cGMP-dependent kinase activation. Such pfPKG versus hPKG rewiring of allosteric networks was unexpected because of the structural similarity between the two essential CBDs. Yet, such finding provides crucial information on which elements to target for selective inhibition of pfPKG versus hPKG, which may potentially reduce undesired side effects in malaria treatments.

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in PfPKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the PfPKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the PfPKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central β-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design PfPKG-selective allosteric inhibitors as a viable treatment for malaria.

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays the most crucial role in PfPKG regulation. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking.

Mechanism of Allosteric Inhibition of Plasmodium falciparum CGMP-Dependent Protein Kinase

Malaria is a life-threatening disease responsible for about a million fatalities per year worldwide. Most of the malaria deaths are caused by Plasmodium falciparum. An essential regulator for the development of P. falciparum is the cyclic GMP (cGMP) dependent protein kinase (PfPKG). PfPKG is composed of a regulatory domain and a catalytic domain. In the absence of cGMP, the regulatory domain inhibits the kinase domain. Upon cGMP binding to the regulatory domain, the inhibition is released and PfPKG is activated. Targeting directly the active site of PfPKG poses a major selectivity challenge, since the kinase catalytic domains are highly conserved among eukaryotes. One approach to circumvent this problem is to selectively target less conserved allosteric sitesof PfPKG, such as the cGMP-binding domains (CBDs), which can be achieved through cGMP-analogs. Here, we report the mechanism of action for cGMP-antagonists of PfPKG to gain structural and dynamical insight on the otherwise elusive bound-inhibited state of PfPKG. This will provide another avenue to rationally design PfPKG-selective inhibitors for the treatment of malaria. NMR Chemical Shift Projection Analysis (CHESPA) of PfPKG CBD-D with cGMP-analogsshows that the cGMP-antagonist inhibits PfPKG not through a simple reversal of a two-state active-inactive equilibrium, but through multi-state equilibria sampling distinct holo-inactive intermediate that combine elements of both active and inactive states in different regions of CBD-D. This intermediate state exhibits inhibitory competent characteristics and also provides the inhibitors the avenue for preserving a high affinity for CBD-D. NMR spin relaxation measurements further complement the CHESPA data by probing the dynamical changes within the CBD-D of PfPKG that occur upon inhibition.

The Molecular Organization of Human cGMP Specific Phosphodiesterase 6 (PDE6): Structural Implications of Somatic Mutations in Cancer and Retinitis Pigmentosa

In the cyclic guanosine monophosphate (cGMP) signaling pathway, phosphodiesterase 6 (PDE6) maintains a critical balance of the intracellular concentration of cGMP by catalyzing it to 5′ guanosine monophosphate (5′-GMP). To gain insight into the mechanistic impacts of the PDE6 somatic mutations that are implicated in cancer and retinitis pigmentosa, we first defined the structure and organization of the human PDE6 heterodimer using computational comparative modelling. Each subunit of PDE6αβ possesses three domains connected through long α-helices. The heterodimer model indicates that the two chains are likely related by a pseudo two-fold axis. The N-terminal region of each subunit is comprised of two allosteric cGMP-binding domains (Gaf-A & Gaf-B), oriented in the same way and interacting with the catalytic domain present at the C-terminal in a way that would allow the allosteric cGMP-binding domains to influence catalytic activity. Subsequently, we applied an integrated knowledge-driven in silico mutation analysis approach to understand the structural and functional implications of experimentally identified mutations that cause various cancers and retinitis pigmentosa, as well as computational saturation mutagenesis of the dimer interface and cGMP-binding residues of both Gaf-A, and the catalytic domains. We studied the impact of mutations on the stability of PDE6αβ structure, subunit-interfaces and Gaf-cGMP interactions. Further, we discussed the changes in interatomic interactions of mutations that are destabilizing in Gaf-A (R93L, V141 M, F162 L), catalytic domain (D600N, F742 L, F776 L) and at the dimer interface (F426A, F248G, F424 N). This study establishes a possible link of change in PDE6αβ structural stability to the experimentally observed disease phenotypes.

Generation of a cGMP Indicator with an Expanded Dynamic Range by Optimization of Amino Acid Linkers between a Fluorescent Protein and PDE5α.

Here we describe the development of a single fluorescent protein (FP)-based cGMP indicator, Green cGull, based on the cGMP binding domain from mouse phosphodiesterase 5α. The dynamic range of Green cGull was enhanced to a 7.5-fold fluorescence change upon cGMP binding by optimization of the amino acid linkers between the cGMP binding domain and FP. Green cGull has excitation and emission peaks at 498 and 522 nm, respectively, and specifically responds to cGMP in a dose-dependent manner. Live cell imaging analysis revealed that addition of a nitric oxide (NO) donor induced different cGMP kinetics and was cell-type dependent. We also found that the NO donor induced an increase of intracellular cGMP, while intracellular Ca2+ exhibited a complex profile, as revealed by dual-color imaging of cGMP and Ca2+. The results suggest that Green cGull sheds new light on understanding the complex interactions between various signaling molecules by multicolor imaging and that our systematic strategy for expanding the dynamic range of single-FP-based indicators is valuable to generate indicators for molecules of interest.

Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein S-Guanylation.

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of guanosine 3',5'-cyclic monophosphate (cGMP) formed endogenously under conditions associated with production of both reactive oxygen species and nitric oxide. It acts as an electrophilic second messenger in the regulation of cellular signaling by inducing a post-translational modification of redox-sensitive protein thiols via covalent adduction of cGMP moieties to protein thiols (protein S-guanylation). Here, we demonstrate that 8-nitro-cGMP potentially S-guanylates thiol groups of cGMP-dependent protein kinase (PKG), the enzyme that serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses. S-Guanylation of PKG was found to occur in a site specific manner; Cys42 and Cys195 were the susceptible residues among 11 Cys residues. Importantly, S-guanylation at Cys195, which is located in the high-affinity cGMP binding domain of PKG, causes persistent enzyme activation as determined by in vitro kinase assay as well as by an organ bath assay. In vivo, S-guanylation of PKG was demonstrated to occur in mice without any specific treatment and was significantly enhanced by lipopolysaccharide administration. These findings warrant further investigation in terms of the physiological and pathophysiological roles of S-guanylation-dependent persistent PKG activation.

Identification of novel mutations by targeted exome sequencing and the genotype-phenotype assessment of patients with achromatopsia.

BackgroundAchromatopsia (ACHM) is a severe congenital autosomal recessive retinal disorder caused by loss of cone photoreceptors. Here, we aimed to determine the underlying genetic lesions and phenotypic correlations in two Chinese families with ACHM.MethodsMedical history and clinical evaluation were obtained from both families. Targeted exome sequencing (TES) was performed on 201 disease-causing genes of inherited retinal dystrophies to screen for ACHM causative mutations in the two probands.ResultsThe compound heterozygous mutations in CNGA3 (c.1074G > A, p.W358X; c.1706G > A, p.R569H) were identified in the first proband, and a novel homozygous mutation (c.968C > A, p.A323D) was detected in the other pedigree. The proposed topological model of the CNGA3 polypeptide suggested that the missense mutations primarily affected the transmembrane helix 5 and the cGMP-binding domain, respectively. Crystal structure modeling of the cyclic nucleotide-gated cation channel α-3 (CNGA3) protein encoded by the CNGA3 gene revealed an abnormal combined structure generated by R569H.ConclusionsWe firstly used the TES approach to identify genetic alterations in patients with ACHM. We uncovered three mutations in CNGA3, including one novel mutation. Our results not only expand the genotypic spectrum for CNGA3 mutations, but also demonstrate that the TES approach is a valuable tool for molecular diagnosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0694-7) contains supplementary material, which is available to authorized users.

Crystal structures of the carboxyl cGMP binding domain of plasmodium falciparumcGMP-dependent protein kinase reveals a novel salt bridge crucial for activation

Background Plasmodium falciparum cGMP-dependent protein kinase (pfPKG) is a validated therapeutic target of malaria. As a key regulator of its life cycle, pfPKG plays a crucial role in both the sexual and asexual blood-stages that cause malaria pathology. Inhibiting pfPKG blocks proliferation and transmission of the parasite [1,2]. However the development of pfPKG-specific inhibitor has been greatly hampered by the lack of high-resolution structure information to guide drug design. Targeting the ATP binding site of pfPKG is an approach commonly associated with low specificity and toxicity [3]. Therefore, we aim to target a domain that is unique to this kinase, the cyclic nucleotide binding (CNB) domain. Since previous studies demonstrated the fourth-cyclic nucleotide binding (CNB-D) domain of pfPKG to be the most important for the kinase activation [4] we focused on this domain to understand its role in cGMP dependent activation.

Crystal Structure of cGMP-Dependent Protein Kinase Reveals Novel Site of Interchain Communication

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 2.5Å crystal structure of a regulatory domain construct (aa 78-355) containing both cGMP binding sites of PKG Iα. 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.

Abstract 5443: A novel biosensor for monitoring intracellular cGMP in live cells

Abstract The second messenger, cyclic guanosine 3’, 5’-monophosphate (cGMP) is a critical and multifunctional signaling molecule involved in many cellular processes. Intracellular cGMP is synthesized by guanylyl cyclase (GC) and is degraded by various phosphodiesterase (PDE) isozymes. Increased levels of cGMP can activate cGMP-dependent protein kinase (PKG), modulate cGMP-gated ion channels, or affect PDE activity. Since cGMP plays an important physiological role and may also be involved in various pathological conditions, including cancer, CNS disorders, and vascular deficiencies, cGMP-specific PDE isozymes represent an important class of drug targets. In order to measure intracellular cGMP levels in live cells, we constructed a novel genetically encoded cGMP biosensor with cGMP binding domains (from human PDE5A GAF-A domain) fused to a modified firefly luciferase. A lipid based reagent was used to transiently transfect HEK293 with this biosensor. Cells were then treated with the nitric oxide donors, sodium nitroprusside (SNP) or NOR-3, which are known to activate soluble GC. Bioluminescence was measured every 2 minutes for at least 60 minutes following treatment. Both SNP (50 µM) and NOR-3 (100 µM) caused a 3-fold increase in bioluminescence compared with vehicle treated cells, reaching a maximum within 10 to 20 minutes of compound addition. Co-transfection with soluble GC further increased the response up to 13-fold following SNP treatment. In addition, PDE inhibitors such as, zaprinast, dipyridamole and 3-isobutyl-1-methylxanthine amplified the response to SNP treatment. As expected, co-transfection with PDE5A decreased the basal level of cGMP and increased the sensitivity of the cGMP response to zaprinast and SNP, as well as the combination. These results suggest that this cGMP biosensor can be useful for studying drug effects on cGMP signaling in live cells. Methods to use the assay for high throughput screening are also being developed. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5443. doi:10.1158/1538-7445.AM2011-5443

Abstract 4610: NO-NSAIDs inhibit colon tumor cell growth by a cGMP-independent mechanism

Abstract Nitric oxide-releasing NSAIDs (NO-NSAIDs) were originally developed as prodrugs to reduce the gastrointestinal toxicity of conventional NSAIDs and were found to have improved potency to inhibit tumor growth compared with the parent NSAID. Although different mechanisms have been proposed for their potent anticancer properties, the role of the NO-moiety has not been clearly defined. We have investigated whether the NO group contributes to the anticancer properties of NO-NSAIDs by inducing soluble guanylyl cyclase and elevating intracellular cGMP levels, effects that are associated with the activity of NO donors. We confirmed the increased potency of the NO-derivatives of sulindac, aspirin (o-, p- and m- isomers), exisulind and naproxen with respect to the parent compounds for inhibiting growth of HCT116, SW480 and COLO741 human colon tumor cells. The two derivatives with the lowest IC50 values, NO-sulindac (16.50 µM) and NO-ASA(o) (8 µM) were also tested for cGMP elevation using a novel cGMP biosensor assay in live cells. In brief, HEK293 cells were transfected with a biosensor construct containing cGMP binding domains fused to the modified firefly luciferase gene (Promega) which produces bioluminescence in the cGMP-bound or ‘activated’ state. Cells were treated with NO-Sulindac, NO-ASA(o) and known NO-donors, NOR-3 and SNP as positive controls. Chemiluminescence was measured over a period of 1h. A strong and sustained signal was obtained with the NO-donors, SNP and NOR-3 (50 µM), although no induction was observed with NO-NSAID treatment (50 µM). In addition, SW480 colon tumor cells were treated with 50 µM NO-Sulindac, NO-ASA(o) or NOR-3 and levels of phosphorylated vasoactivator stimulated phosphoproteins (p-VASP), an intracellular marker of cGMP signaling activity, were measured by Western Blotting. NOR-3 caused a time-dependent increase in p-VASP expression, whereas the NO-NSAIDs had no such effect. Since sulindac and certain derivatives have previously been reported to inhibit cGMP-phosphodiesterase (PDE) and inhibit tumor cell growth by a mechanism involving cGMP elevation, we also determined if NO-NSAIDS can inhibit cGMP PDE activity. NO-NSAIDs did not inhibit cGMP hydrolysis by either recombinant PDE5 or in lysates from colon tumor cells, although sulindac sulfide was an effective inhibitor as previously reported. These results suggest that mechanisms other than cGMP elevation explain the anticancer properties of NO-NSAIDs. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2011-4610

The amino terminus of cGMP-dependent protein kinase Iβ increases the dynamics of the protein's cGMP-binding pockets

The type I cGMP-dependent protein kinases play critical roles in regulating vascular tone, platelet activation and synaptic plasticity. PKG Iα and PKG Iβ 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 Iα has a >10-fold higher affinity for cGMP than PKG Iβ, and PKG Iβ that is missing its leucine zipper has a 3-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 Iβ’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.

A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with “detector” proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2′-O-(N-methylanthraniloyl) guanosine 5′-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Small-molecule modulators of cGMP signaling are of interest to basic and clinical research. The cGMP-dependent protein kinase type I (cGKI) is presumably a major mediator of cGMP effects, and the cGMP analogue Rp-8-Br-PET-cGMPS (Rp-PET) (chemical name: beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer) is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies. Here, we have evaluated the properties of Rp-PET using cGKI-expressing and cGKI-deficient primary vascular smooth muscle cells (VSMCs), purified cGKI isozymes, and an engineered cGMP sensor protein. cGKI activity in intact VSMCs was monitored by cGMP/cGKI-stimulated cell growth and phosphorylation of vasodilator-stimulated phosphoprotein. Unexpectedly, Rp-PET (100 microm) did not efficiently antagonize activation of cGKI by the agonist 8-Br-cGMP (100 microm) in intact VSMCs. Moreover, in the absence of 8-Br-cGMP, Rp-PET (100 microm) stimulated cell growth in a cGKIalpha-dependent manner. Kinase assays with purified cGKI isozymes confirmed the previously reported inhibition of the cGMP-stimulated enzyme by Rp-PET in vitro. However, in the absence of the agonist cGMP, Rp-PET partially activated the cGKIalpha isoform. Experiments with a fluorescence resonance energy transfer-based construct harboring the cGMP binding sites of cGKI suggested that binding of Rp-PET induces a conformational change similar to the agonist cGMP. Together, these findings indicate that Rp-PET is a partial cGKIalpha agonist that under certain conditions stimulates rather than inhibits cGKI activity in vitro and in intact cells. Data obtained with Rp-PET as cGKI inhibitor should be interpreted with caution and not be used as sole evidence to dissect the role of cGKI in signaling processes.

Intramolecular Activation Mechanism of the Dictyostelium LRRK2 Homolog Roco Protein GbpC

GbpC is a large multidomain protein involved in cGMP-mediated chemotaxis in the cellular slime mold Dictyostelium discoideum. GbpC belongs to the Roco family of proteins that often share a central core region, consisting of leucine-rich repeats, a Ras domain (Roc), a Cor domain, and a MAPKKKinase domain. In addition to this core, GbpC contains a RasGEF domain and two cGMP-binding domains. Here, we report on an intramolecular signaling cascade of GbpC. In vitro, the RasGEF domain of GbpC specifically accelerates the GDP/GTP exchange of the Roc domain. Moreover, cGMP binding to GbpC strongly stimulates the binding of GbpC to GTP-agarose, suggesting cGMP-stimulated GDP/GTP exchange at the Roc domain. The function of the protein in vivo was investigated by rescue analysis of the chemotactic defect of gbpC null cells. Mutants that lack a functional guanine exchange factor (GEF), Roc, or kinase domain are inactive in vivo. Together, the results suggest a four-step intramolecular activation mechanism of the Roco protein GbpC: cGMP binding to the cyclic nucleotide-binding domains, activation of the GEF domain, GDP/GTP exchange of Roc, and activation of the MAPKKK domain.

CGMP-binding Prepares PKG for Substrate Binding by Disclosing the C-terminal Domain

Type I cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase (PKG) is involved in the nitric oxide/cGMP signaling pathway. PKG has been identified in many different species, ranging from unicelõlular organisms to mammals. The enzyme serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses, ranging from smooth-muscle relaxation to neuronal synaptic plasticity. In the absence of a crystal structure, the three-dimensional structure of the homodimeric 152-kDa kinase PKG is unknown; however, there is evidence that the kinase adopts a distinct cGMP-dependent active conformation when compared to the inactive conformation. We performed mass-spectrometry-based hydrogen/deuterium exchange experiments to obtain detailed information on the structural changes in PKG Iα induced by cGMP activation. Site-specific exchange measurements confirmed that the autoinhibitory domain and the hinge region become more solvent exposed, whereas the cGMP-binding domains become more protected in holo-PKG (dimeric PKG saturated with four cGMP molecules bound). More surprisingly, our data revealed a specific disclosure of the substrate-binding region of holo-PKG, shedding new light into the kinase-activation process of PKG.

Cyclic GMP-gated channel and peripherin/rds-rom-1 complex of rod cells.

The cGMP-gated channel and the peripherin/rds-rom-1 complex are two oligomeric membrane proteins that play key roles in the structure and function of photoreceptor outer segments. The channel is localized on the plasma membrane where it controls the flow of Na+ and Ca2+ into the outer segment in response to light-induced changes in cGMP. The rod channel consists of two homologous subunits, designated alpha and beta, which assemble into a heterotetrameric complex. Both subunits contain a core structural unit consisting of six transmembrane segments, a pore region and a cGMP binding domain. The alpha subunit is the dominant functional subunit since it forms a functional channel by itself. The beta subunit does not assemble into a functional channel by itself, but modulates the activity of the channel. The peripherin/rds-rom-1 complex is localized along the rim region of disk membranes where it plays a crucial role in disk morphogenesis. This complex consists of two peripherin/rds and two rom-1 subunits that interact non-covalently to form a heterotetramer. Peripherin/rds is the dominant subunit since, in the absence of rom-1, it self-assembles into a homotetramer that effectively supports outer segment disk formation and structure. Rom-1 on its own does not initiate outer segment formation. Instead, it plays a minor role in fine tuning disk structure. Recently, peripherin/rds-containing tetramers have been shown to form disulfide-mediated higher-order oligomers. This novel oligomerization is suggested to play a central role in outer segment disk formation.

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.