Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R.

In murine and bovine photoreceptors, guanylate cyclase-activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase-activating protein 1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg2+ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, nonmyristoylated GCAP2 does not bind Mg2+ over the physiological range and remains as a monomer in the absence of Ca2+. Both myristoylated and nonmyristoylated GCAP2 bind Ca2+ with high affinity. At odds with guanylate cyclase-activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD.

Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase.

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase-activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone-rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone-rod dystrophy 6 pathology.

Retinal degeneration-3 protein promotes photoreceptor survival by suppressing activation of guanylyl cyclase rather than accelerating GMP recycling.

Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the rd3 mouse strain and in human Leber's congenital amaurosis, a congenital retinal dystrophy that results in early vision loss. However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3/rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.

Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa.

The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G>C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca2+-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 ms molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca2+ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.

GAPDH delivers heme to soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key component of NO-cGMP signaling in mammals. Although heme must bind in the sGC ß1 subunit (sGCß) for sGC to function, how heme is delivered to sGCß remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCß involves GAPDH. We utilized an sGCß reporter construct, tetra-Cys sGCß, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCß in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCß correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCß associates with GAPDH in cells and dissociates when heme binds sGCß, and that the purified GAPDH-heme complex binds to apo-sGCß and transfers its heme to sGCß. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCß to accomplish heme delivery to sGCß. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.

Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase.

Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase-activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K-AKT-nNOS signaling.

Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.

GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone-rod dystrophy but not for stationary night blindness.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone-rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer-6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Retinal degeneration 3 (RD3) protein, a retinal guanylyl cyclase regulator, forms a monomeric and elongated four-helix bundle.

Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in the photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase-activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18-160) that is monomeric and can still bind and negatively regulate RetGC. The NMR solution structure of RD3 revealed an elongated backbone structure (70 Å long and 30 Å wide) consisting of a four-helix bundle with a long unstructured loop between helices 1 and 2. The structure reveals that RD3 residues previously implicated in the RetGC binding map to a localized and contiguous area on the structure, involving a loop between helices 2 and 3 and adjacent parts of helices 3 and 4. The NMR structure of RD3 was validated by mutagenesis. Introducing Trp85 or Phe29 to replace Cys or Leu, respectively, disrupts packing in the hydrophobic core and lowers RD3's apparent affinity for RetGC1. Introducing a positive charge at the interface (Glu32 to Lys) also lowered the affinity. Conversely, introducing Val in place of Cys93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase. The NMR structure of RD3 presented here provides a structural basis for elucidating RD3-RetGC interactions relevant for normal vision or blindness.

Retinal guanylyl cyclase activation by calcium sensor proteins mediates photoreceptor degeneration in an rd3 mouse model of congenital human blindness.

Deficiency of RD3 (retinal degeneration 3) protein causes recessive blindness and photoreceptor degeneration in humans and in the rd3 mouse strain, but the disease mechanism is unclear. Here, we present evidence that RD3 protects photoreceptors from degeneration by competing with guanylyl cyclase-activating proteins (GCAPs), which are calcium sensor proteins for retinal membrane guanylyl cyclase (RetGC). RetGC activity in rd3/rd3 retinas was drastically reduced but stimulated by the endogenous GCAPs at low Ca2+ concentrations. RetGC activity completely failed to accelerate in rd3/rd3GCAPs-/- hybrid photoreceptors, whose photoresponses remained drastically suppressed compared with the WT. However, ∼70% of the hybrid rd3/rd3GCAPs-/- photoreceptors survived past 6 months, in stark contrast to <5% in the nonhybrid rd3/rd3 retinas. GFP-tagged human RD3 inhibited GCAP-dependent activation of RetGC in vitro similarly to the untagged RD3. When transgenically expressed in rd3/rd3 mouse retinas under control of the rhodopsin promoter, the RD3GFP construct increased RetGC levels to near normal levels, restored dark-adapted photoresponses, and rescued rods from degeneration. The fluorescence of RD3GFP in rd3/rd3RD3GFP+ retinas was mostly restricted to the rod photoreceptor inner segments, whereas GCAP1 immunofluorescence was concentrated predominantly in the outer segment. However, RD3GFP became distributed to the outer segments when bred into a GCAPs-/- genetic background. These results support the hypothesis that an essential biological function of RD3 is competition with GCAPs that inhibits premature cyclase activation in the inner segment. Our findings also indicate that the fast rate of degeneration in RD3-deficient photoreceptors results from the lack of this inhibition.

Synergistic mutations in soluble guanylyl cyclase (sGC) reveal a key role for interfacial regions in the sGC activation mechanism.

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) and a central component of the NO-cGMP pathway, critical to cardiovascular function. NO binding to the N-terminal sensor domain in sGC enhances the cyclase activity of the C-terminal catalytic domain. Our understanding of the structural elements regulating this signaling cascade is limited, hindering structure-based drug design efforts that target sGC to improve the management of cardiovascular diseases. Conformational changes are thought to propagate the NO-binding signal throughout the entire sGC heterodimer, via its coiled-coil domain, to reorient the catalytic domain into an active conformation. To identify the structural elements involved in this signal transduction cascade, here we optimized a cGMP-based luciferase assay that reports on heterologous sGC activity in Escherichia coli and identified several mutations that activate sGC. These mutations resided in the dorsal flaps, dimer interface, and GTP-binding regions of the catalytic domain. Combinations of mutations from these different elements synergized, resulting in even greater activity and indicating a complex cross-talk among these regions. Molecular dynamics simulations further revealed conformational changes underlying the functional impact of these mutations. We propose that the interfacial residues play a central role in the sGC activation mechanism by coupling the coiled-coil domain to the active site via a series of hot spots. Our results provide new mechanistic insights not only into the molecular pathway for sGC activation but also for other members of the larger nucleotidyl cyclase family.

A G86R mutation in the calcium-sensor protein GCAP1 alters regulation of retinal guanylyl cyclase and causes dominant cone-rod degeneration.

The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca2+ concentrations decline, and decelerates the cyclase in the dark, when Ca2+ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca2+-dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca2+ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca2+ concentrations characteristic of dark-adapted photoreceptors. Ca2+-dependent increase in Trp94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca2+ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca2+, and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca2+ sensitivity of the cyclase within the physiological range of intracellular Ca2+ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly86, leading to abnormally high affinity for the target enzyme and reduced Ca2+ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca2+ influx in photoreceptors in the dark.

Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties.

Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the ß1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

Guanylate cyclase-activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors.

Light adaptation of photoreceptor cells is mediated by Ca2+-dependent mechanisms. In darkness, Ca2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca2+ extrusion via Na+/Ca2+, K+ exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca2+ levels in photoreceptor outer segment because of continuing Ca2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca2+ This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca2+-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca2+ when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

Guanylyl cyclase sensitivity to nitric oxide is protected by a thiol oxidation-driven interaction with thioredoxin-1.

Nitric oxide (NO) modulates many physiological events through production of cGMP from its receptor, the NO-sensitive guanylyl cyclase (GC1). NO also appears to function in a cGMP-independent manner, via S-nitrosation (SNO), a redox-based modification of cysteine thiols. Previously, we have shown that S-nitrosated GC1 (SNO-GC1) is desensitized to NO stimulation following prolonged NO exposure or under oxidative/nitrosative stress. In animal models of nitrate tolerance and angiotensin II-induced hypertension, decreased vasodilation in response to NO correlates with GC1 thiol oxidation, but the physiological mechanism that resensitizes GC1 to NO and restores basal activity is unknown. Because GC1 interacts with the oxidoreductase protein-disulfide isomerase, we hypothesized that thioredoxin-1 (Trx1), a cytosolic oxidoreductase, could be involved in restoring GC1 basal activity and NO sensitivity because the Trx/thioredoxin reductase (TrxR) system maintains thiol redox homeostasis. Here, by manipulating activity and levels of the Trx1/TrxR system and by using a Trx1-Trap assay, we demonstrate that Trx1 modulates cGMP synthesis through an association between Trx1 and GC1 via a mixed disulfide. A proximity ligation assay confirmed the endogenous Trx1-GC1 complex in cells. Mutational analysis suggested that Cys609 in GC1 is involved in the Trx1-GC1 association and modulation of GC1 activity. Functionally, we established that Trx1 protects GC1 from S-nitrosocysteine-induced desensitization. A computational model of Trx1-GC1 interaction illustrates a possible mechanism for Trx1 to maintain basal GC1 activity and prevent/rescue GC1 desensitization to NO. The etiology of some oxidative vascular diseases may very well be explained by the dysfunction of the Trx1-GC1 association.

Skeletal overgrowth-causing mutations mimic an allosterically activated conformation of guanylyl cyclase-B that is inhibited by 2,4,6,-trinitrophenyl ATP.

Activating mutations in the receptor for C-type natriuretic peptide (CNP), guanylyl cyclase B (GC-B, also known as Npr2 or NPR-B), increase cellular cGMP and cause skeletal overgrowth, but how these mutations affect GTP catalysis is poorly understood. The A488P and R655C mutations were compared with the known mutation V883M. Neither mutation affected GC-B concentrations. The A488P mutation decreased the EC50 5-fold, increased Vmax 2.6-fold, and decreased the Km 13-fold, whereas the R655C mutation decreased the EC50 5-fold, increased the Vmax 2.1-fold, and decreased the Km 4.7-fold. Neither mutation affected maximum activity at saturating CNP concentrations. Activation by R655C did not require disulfide bond formation. Surprisingly, the A488P mutant only activated the receptor when it was phosphorylated. In contrast, the R655C mutation converted GC-B-7A from CNP-unresponsive to CNP-responsive. Interestingly, neither mutant was activated by ATP, and the Km and Hill coefficient of each mutant assayed in the absence of ATP were similar to those of wild-type GC-B assayed in the presence of ATP. Finally, 1 mm 2,4,6,-trinitrophenyl ATP inhibited all three mutants by as much as 80% but failed to inhibit WT-GC-B. We conclude that 1) the A488P and R655C missense mutations result in a GC-B conformation that mimics the allosterically activated conformation, 2) GC-B phosphorylation is required for CNP-dependent activation by the A488P mutation, 3) the R655C mutation abrogates the need for phosphorylation in receptor activation, and 4) an ATP analog selectively inhibits the GC-B mutants, indicating that a pharmacologic approach could reduce GC-B dependent human skeletal overgrowth.

Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC.

RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCCRho) and without (GCRho) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GCRho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GCRho monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein.

Retinal degeneration 3 (RD3) protein, essential for normal expression of retinal membrane guanylyl cyclase (RetGC) in photoreceptor cells, blocks RetGC catalytic activity and stimulation by guanylyl cyclase-activating proteins (GCAPs). In a mouse retina, RD3 inhibited both RetGC1 and RetGC2 isozymes. Photoreceptors in the rd3/rd3 mouse retinas lacking functional RD3 degenerated more severely than in the retinas lacking both RetGC isozymes, consistent with a hypothesis that the inhibitory activity of RD3 has a functional role in photoreceptors. To map the potential target-binding site(s) on RD3, short evolutionary conserved regions of its primary structure were scrambled and the mutations were tested for the RD3 ability to inhibit RetGC1 and co-localize with the cyclase in co-transfected cells. Substitutions in 4 out of 22 tested regions, (87)KIHP(90), (93)CGPAI(97), (99)RFRQ(102), and (119)RSVL(122), reduced the RD3 apparent affinity for the cyclase 180-700-fold. Changes of amino acid sequences outside the Lys(87)-Leu(122) central portion of the molecule either failed to prevent RD3 binding to the cyclase or had a much smaller effect. Mutations in the (93)CGPAI(97) portion of a predicted central α-helix most drastically suppressed the inhibitory activity of RD3 and disrupted RD3 co-localization with RetGC1 in HEK293 cells. Different side chains replacing Cys(93) profoundly reduced RD3 affinity for the cyclase, irrespective of their relative helix propensities. We conclude that the main RetGC-binding interface on RD3 required for the negative regulation of the cyclase localizes to the Lys(87)-Leu(122) region.