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Nitric oxide (NO) affects many physiological systems by activating cGMP signaling cascades through soluble guanylate cyclase (sGC). In the accepted model, NO binds to the sGC heme, activating the enzyme. Here, we report that in the presence of physiological concentrations of ATP and GTP, NO dissociation from the sGC heme is Ϸ160 times slower than the rate of enzyme deactivation in vitro. Deactivated sGC still has NO bound to the heme, and full activation requires additional NO. We propose an activation model where, in the presence of both ATP and GTP, tonic NO forms a stable heme complex with low sGC activity; acute production of NO transiently and fully activates this NO-bound sGC.heme ͉ nucleotide regulation N itric oxide (NO) mediates blood vessel relaxation, complex aspects of myocardial function, perfusion and function of all major organs, synaptic plasticity in the brain, platelet aggregation, skin function, and numerous other physiological processes, by targeting and activating soluble guanylate cyclase (sGC) (reviewed in refs. 1-5). Dysregulation of NO signaling, then, contributes to many types of disease state, from erectile dysfunction and heart disease, to neurodegeneration, stroke, hypertension, and gastrointestinal disease, to name a few (reviewed in refs. 6-8).In vivo and ex vivo tissue studies of NO have revealed two fundamentally distinct and paradoxical signaling modes: tonic and acute. Tonic NO describes the continual low-level production of NO that elicits a long-lasting low-level cGMP signal (9). Under resting conditions such as normotension, inhibition of nitric oxide synthase (NOS) or sGC results in vasoconstriction; therefore, tonic NO-elicited cGMP production maintains homeostatic vascular tone. Relaxation of smooth muscle requires an acute burst of NO synthesis, typically triggered by acetylcholine, and cGMP levels rise rapidly (10). These data, which describe two separate effects of NO, cannot be explained by the established binary model for NO regulation of sGC: that NO activates sGC solely by binding to its heme cofactor.The N-terminal Ϸ180 aa of the ␤1 subunit of sGC form an evolutionarily conserved protoporphyrin-IX heme domain with spectral properties similar to the holoenzyme (11-13). NO binding to the sGC heme is diffusion-limited; however, oxygen does not bind to sGC, and carbon monoxide (CO) binding is at least 10 6 -fold weaker than NO. Thus, the sGC heme environment is a specific NO sensor. The C-terminal domains of each subunit are homologous to the catalytic domains of adenylate cyclase and fold together to form the active site of the enzyme (14). In the existing activation model (Fig. 1, black scheme), NO binds to the heme of sGC (15), forming a six-coordinate intermediate (16)(17)(18). The rate of conversion of this intermediate to the final five-coordinate ferrous-nitrosyl species depends on the concentration of NO; thus, nonheme NO accelerates rupture of the proximal histidine-iron bond. Breaking of this bond is thought to result in a conformational change in the cataly...

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.

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.

Background: Imatinib represents the first in a class of drugs targeted against chronic myelogenous leukemia to enter the clinic, showing excellent efficacy and specificity for Abl, Kit, and PDGFR kinases. Recent screens carried out to find off-target proteins that bind to imatinib identified the oxidoreductase NQO2, a flavoprotein that is phosphorylated in a chronic myelogenous leukemia cell line.

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length ␣1␤1 sGC, and the constructs ␤1(1-194), ␤1(1-385), and ␤2(1-217), at 37 and 10°C with and without the substrate analogue guanosine-5-[(␣,␤-methylene]triphosphate (GMPCPP) or the activator 3-(5-hydroxymethyl-3-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and ␤2(1-217) at 37°C and dramatically accelerated NO dissociation from sGC at 10°C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10°C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states. Soluble guanylate cyclase (sGC)4 is the best characterized physiological receptor for the gaseous signaling agent nitric oxide (NO) (1-5). In response to NO, sGC produces the second messenger cGMP, modulating physiological processes such as neurotransmission and vasodilation (6). The ␣1␤1 sGC heterodimer is activated several hundredfold above the basal level by the binding of NO to the heme of the ␤1 H-NOX domain (7-9), a conserved domain of unique structure (10 -12). However, it remains unclear how this binding event is translated into increased catalytic activity.The mechanism by which sGC deactivation occurs has been a focus of much investigation (8,13,14). Initially thought to result from simple dissociation of NO from the heme, the deactivation process has turned out to be more complicated. In fact, regulation of sGC by NO has been shown to involve a complex interplay between binding of NO to the heme and to non-heme sites as well as allosteric regulation by GTP, also a substrate, and ATP, a reporter for the energy status of the cell (15). In integrating the inputs from all these signals, sGC has been shown to have a remarkable attribute, the ability to exist in a stable low activity state or a transient high activity state, both containing NO bound to the heme (16, 17). Many questions concerning the existence and characteristics of these two sGC heme-NO species, and how t...

Zeta-chain Associated Protein of 70kDa (ZAP-70) and Spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signaling, respectively. They are recruited, via their tandem-SH2 domains, to doubly-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signaling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys 39 in ZAP-70, Cys 206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of hydrogen peroxide, and these two cysteine residues are also necessary for inhibition by hydrogen peroxide. Our findings suggest a mechanism by which the generation of reactive oxygen species generated during responses to antigen could attenuate signaling through these kinases, and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.

ZAP-70 is a critical molecule in the transduction of T-cell antigen receptor signaling and the activation of T cells. Upon activation of the T-cell antigen receptor, ZAP-70 is recruited to the intracellular ζ chains of the T-cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T-cell antigen receptor. The screen is based on a fluorescence polarization assay for peptide binding to ZAP-70.