The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

Background

Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases.

Results

We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule.

Conclusion

Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.     

The intracellular carboxyl terminal domain of Vangl proteins contains plasma membrane targeting signals

Vangl1 and Vangl2 are integral membrane proteins that play a critical role in establishing planar cell polarity (PCP) in epithelial cells and are required for convergent extension (CE) movements during embryogenesis. Their proper targeting to the plasma membrane (PM) is required for function. We created discrete deletions at the amino and carboxy termini of Vangl1 and monitored the effect of the mutations on PM targeting in Madin–Darby canine kidney cells. Our results show that the Vangl1 amino terminus lacks PM targeting determinants, and these are restricted to the carboxy terminus, including the predicted PDZBM motif at the C‐terminus.     

The membrane form of guanylate cyclase. Homology with a subunit of the cytoplasmic form of the enzyme

A cDNA clone for the membrane form of guanylate cyclase has been isolated from the testis of the sea urchin Strongylocentrotus purpuratus. An open reading frame predicts a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids divided the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl domain of 594 intracellular amino acids. Three potential Asn-linked glycosylation sites were present in the proposed extracellular domain. The deduced protein sequence was homologous to the protein kinase family and contained limited but significant regions of identity with a low molecular weight atrial natriuretic peptide receptor. The carboxyl region (202 amino acids) was 42% identical with a subunit of the cytoplasmic form of guanylate cyclase recently cloned from bovine lung (Koesling, D., Herz, J., Gausepohl, H., Niroomand, F., Hinsch, K.-D., Mulsch, A., Bohme, E., Schultz, G., and Frank, R. (1988) FEBS Lett. 239, 29-34). Therefore, the membrane form of guanylate cyclase is a member of an apparently large family of proteins that includes the low molecular weight atrial natriuretic peptide receptor, the soluble form of guanylate cyclase and protein kinases.     

Probing domain interactions in soluble guanylate cyclase

Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat β1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the β1 H-NOX construct (residues 1-194), the β1 H-NOX-PAS-CC construct (residues 1-385), and the full-length α1β1 sGC heterodimer (β1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.     

Evolution of the membrane guanylate cyclase transduction system

Almost four decades of research in the field of membrane guanylate cyclases is discussed in this review. Primarily, it focuses on the chronological development of the field, recognizes major contributions of the original investigators, corrects certain misplaced facts, and projects its future trend.     

Surface plasmon resonance using the catalytic domain of soluble guanylate cyclase allows the detection of enzyme activators

Soluble Guanylate Cyclase (sGC) is the receptor for the signalling agent nitric oxide (NO) and catalyses the production of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). The enzyme is an attractive drug target for small molecules that act in the cardiovascular and pulmonary systems, and has also shown to be a potential target in neurological disorders. We have discovered that 5-(indazol-3-yl)-1,2,4-oxadiazoles activate the enzyme in the absence of added NO and shown they bind to the catalytic domain of the enzyme after development of a surface plasmon resonance assay that allows the biophysical detection of intrinsic binding of ligands to the full length sGC and to a construct of the catalytic domain.     

Expression and characterization of the catalytic domains of soluble guanylate cyclase: interaction with the heme domain

The catalytic domains (alpha(cat) and beta(cat)) of alpha1beta1 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. alpha(cat), beta(cat), and the alpha(cat)beta(cat) heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. Alpha(cat) and beta(cat) were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when alpha(cat) and beta(cat) were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full-length enzyme, the alpha(cat)beta(cat) complex was more active in the presence of Mn(2+) as compared to the physiological cofactor Mg(2+), although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The K(M) for Mn(2+)-GTP was measured to be 85 +/- 18 microM, and in the presence of Mn(2+)-GTP, the K(D) for the alpha(cat)beta(cat) complex was 450 +/- 70 nM. The N-terminal heme-bound regulatory domain of the beta1 subunit of sGC inhibited the activity of the alpha(cat)beta(cat) complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed.     

Purification and properties of the phosphorylated form of guanylate cyclase

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.     

The calcium-sensor guanylate cyclase activating protein type 2 specific site in rod outer segment membrane guanylate cyclase type 1

The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.     

Odorant-linked ROS-GC subfamily membrane guanylate cyclase transduction system

This review focuses on the principles of the Ca²⁺-modulated ROS-GC subfamily transduction system linked with the mammalian olfactory transduction field, its historical development, and the present day status on its constitution and operational mechanisms controlling the process of olfactory-transduction. Beginning parts of this article are freely borrowed from the earlier reviews of the authors (Sharma RK, Duda T, Venkataraman V, Koch KW, Curr Topics Biochem Res 6:111–144, 2004; Duda T, Venkataraman V, Sharma RK, Neuronal calcium sensor proteins, pp 91–113, Nova Science Publishers, Inc., 2007).     

An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region

We have identified an integral membrane protein of 145 kD (estimated by SDS-PAGE) of rat liver nuclear envelopes that binds to WGA. We obtained peptide sequence from purified p145 and cloned and sequenced several cDNA clones and one genomic clone. The relative molecular mass of p145 calculated from its complete, cDNA deduced primary structure is 120.7 kD. Antibodies raised against a synthetic peptide represented in p145 reacted monospecifically with p145. In indirect immunofluorescence these antibodies gave punctate staining of the nuclear envelope. Immunogold EM showed specific decoration of the nuclear pores. Thus p145 is an integral membrane protein located specifically in the "pore membrane" domain of the nuclear envelope. To indicate this specific location, and based on its calculated relative molecular mass, the protein is termed POM 121 (pore membrane protein of 121 kD). The 1,199- residue-long primary structure shows a hydrophobic region (residues 29- 72) that is likely to form one (or two adjacent) transmembrane segment(s). The bulk of the protein (residues 73-1199) is predicted to be exposed not on the cisternal side but on the pore side of the pore membrane. It contains 36 consensus sites for various kinases. However, its most striking feature is a repetitive pentapeptide motif XFXFG that has also been shown to occur in several nucleoporins. This nucleoporin- like domain of POM 121 is proposed to function in anchoring components of the nuclear pore complex to the pore membrane.     

Calcium-modulated guanylate cyclase transduction machinery in the photoreceptor--bipolar synaptic region

Rod outer segment membrane guanylate cyclase (ROS-GC) transduction system is a central component of the Ca(2+)-sensitive phototransduction machinery. The system is composed of two parts: Ca(2+) sensor guanylate cyclase activating protein (GCAP) and ROS-GC. GCAP senses Ca(2+) impulses and inhibits the cyclase. This operational feature of the cyclase is considered to be unique and exclusive in the phototransduction machinery. A combination of reconstitution, peptide competition, cross-linking, and immunocytochemical studies has been used in this study to show that the GCAP1/ROS-GC1 transduction system also exists in the photoreceptor synaptic (presynaptic) termini. Thus, the presence of this system and its linkage is not unique to the phototransduction machinery. A recent study has demonstrated that the photoreceptor-bipolar synaptic region also contains a Ca(2+)-stimulated ROS-GC1 transduction system [Duda, T., et al. (2002) EMBO J. 21, 2547-2556]. In this case, S100beta senses Ca(2+) and stimulates the cyclase. The inhibitory and stimulatory Ca(2+)-modulated ROS-GC1 sites are distinct. These findings allow the formation of a new topographic model of ROS-GC1 transduction. In this model, the catalytic module of ROS-GC1 at its opposite ends is flanked by GCAP1 and S100beta modules. GCAP1 senses the Ca(2+) impulse and inhibits the catalytic module; S100beta senses the impulse and stimulates the catalytic module. Thus, ROS-GC1 acts as a bimodal Ca(2+) signal transduction switch in the photoreceptor bipolar synapse.     

The carboxyl terminus of RNA helicase A contains a bidirectional nuclear transport domain

Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathione S-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1.     

Ca2+-modulated membrane guanylate cyclase in the testes

To date, the calcium-regulated membrane guanylate cyclase Rod Outer Segment Guanylate Cyclase type 1 (ROS-GC1) transduction system in addition to photoreceptors is known to be expressed in three other types of neuronal cells: in the pinealocytes, mitral cells of the olfactory bulb and the gustatory epithelium of tongue. Very recent studies from our laboratory show that expression of ROS-GC1 is not restricted to the neuronal cells; the male gonads and the spermatozoa also express ROS-GC1. In this presentation, the authors review the existing information on the localization and function of guanylate cyclase with special emphasis on Ca²⁺-modulated membrane guanylate cyclase, ROS-GC1, in the testes. The role of ROS-GC1 and its Ca²⁺-sensing modulators in the processes of spermatogenesis and fertilization are discussed.     

Plasma membrane guanylate cyclase is a multimodule transduction system

Summary This minireview highlights the studies which suggest that guanylate cyclase is a single-component transducing system, containing distinct signaling modules in a single membrane-spanning protein. A guanylate cyclase signaling model is proposed which envisions the following sequential events: (1) a signal is initiated by the binding of the hormone to the ligand binding module; (2) the signal is potentiated by ATP at ARM; and (3) the amplified signal is finally transduced at the catalytic site. All of these signaling steps together constitute a switch, which when turned on, generates the second messenger cyclic GMP.     

Computational exploration of the binding mode of heme‐dependent stimulators into the active catalytic domain of soluble guanylate cyclase

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41‐2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme‐dependent stimulators and heme‐independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H‐NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an “in silico” approach. Homology models of the catalytic domain of sGC in “inactive” or “active” conformations were constructed using the structure of previously described crystals of the catalytic domains of “inactive” sGCs (2WZ1, 3ET6) and of “active” adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 μs. Docking of YC‐1, a classic heme‐dependent stimulator, to all frames of representative trajectories of “inactive” and “active” conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high‐affinity binding site on the “active” structure. The site, located between the pseudo‐symmetric and the catalytic site just over the loop β₂–β₃, does not overlap with the forskolin binding site on adenylate cyclases. Proteins 2016; 84:1534–1548. © 2016 Wiley Periodicals, Inc.     

The adenylate cyclase catalytic domain of Streptomyces coelicolor is carboxy-terminal

Abstract A DNA fragment of Streptomyces coelicolor encoding the carboxy-terminal catalytic domain of adenylate cyclase was cloned, sequenced and expressed in an Escherichia coli cya -defective strain where it produced nanomole levels of cAMP. The amino acid sequence of the enzyme displays similarities with the Brevibacterium liquefaciens pyruvate regulated adenylate cyclase.     

Differential Ca(2+) sensor guanylate cyclase activating protein modes of photoreceptor rod outer segment membrane guanylate cyclase signaling

Photoreceptor ROS-GC1 (rod outer segment membrane guanylate cyclase) is a vital component of phototransduction. It is a bimodal Ca(2+) signal transduction switch, operating between 20 and ～1000 nM. Modulated by Ca(2+) sensors guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2, respectively), decreasing [Ca(2+)](i) from 200 to 20 nM progressively turns it "on", as does the modulation by the Ca(2+) sensor S100B, increasing [Ca(2+)](i) from 100 to 1000 nM. The GCAP mode plays a vital role in phototransduction in both rods and cones and the S100B mode in the transmission of neural signals to cone ON-bipolar cells. Through a programmed domain deletion, expression, in vivo fluorescence spectroscopy, and in vitro reconstitution experiments, this study demonstrates that the biochemical mechanisms modulated by two GCAPs in Ca(2+) signaling of ROS-GC1 activity are totally different. (1) They involve different structural domains of ROS-GC1. (2) Their signal migratory pathways are opposite: GCAP1 downstream and GCAP2 upstream. (3) Importantly, the isolated catalytic domain, translating the GCAP-modulated Ca(2+) signal into the generation of cyclic GMP, in vivo, exists as a homodimer, the two subunits existing in an antiparallel conformation. Furthermore, the findings demonstrate that the N-terminally placed signaling helix domain is not required for the catalytic domain's dimeric state. The upstream GCAP2-modulated pathway is the first of its kind to be observed for any member of the membrane guanylate cyclase family. It defines a new model of Ca(2+) signal transduction.