Computational exploration of the binding mode of the heme-dependent activator YC-1 into the active catalytic site of soluble guanylate cyclase
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ABSTRACT:
SUBMITTER: Agullo L
PROVIDER: S-EPMC4565073 | biostudies-literature | 2015 Jan
REPOSITORIES: biostudies-literature
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Project description:Soluble guanylate cyclase (sGC) is the principal target of nitric oxide (NO) to control gastrointestinal motility. The consequence on nitrergic signaling and gut motility of inducing a heme-free status of sGC, as induced by oxidative stress, was investigated.sGC?1 (H105F) knock-in (apo-sGC) mice, which express heme-free sGC that has basal activity, but cannot be stimulated by NO, were generated.Diethylenetriamine NONOate did not increase sGC activity in gastrointestinal tissue of apo-sGC mice. Exogenous NO did not induce relaxation in fundic, jejunal and colonic strips, and pyloric rings of apo-sGC mice. The stomach was enlarged in apo-sGC mice with hypertrophy of the muscularis externa of the fundus and pylorus. In addition, gastric emptying and intestinal transit were delayed and whole-gut transit time was increased in the apo-sGC mice, while distal colonic transit time was maintained. The nitrergic relaxant responses to electrical field stimulation at 1-4 Hz were abolished in fundic and jejunal strips from apo-sGC mice, but in pyloric rings and colonic strips, only the response at 1 Hz was abolished, indicating the contribution of other transmitters than NO.The results indicate that the gastrointestinal consequences of switching from a native sGC to a heme-free sGC, which cannot be stimulated by NO, are most pronounced at the level of the stomach establishing a pivotal role of the activation of sGC by NO in normal gastric functioning. In addition, delayed intestinal transit was observed, indicating that nitrergic activation of sGC also plays a role in the lower gastrointestinal tract.
| S-EPMC4932850 | biostudies-literature
Project description:Soluble guanylate cyclase (sGC) is weakly activated by carbon monoxide (CO) but is significantly activated by the binding of YC-1 to the sGC-CO complex. In this report, resonance Raman (RR) spectroscopy was used to study selected sGC variants. Addition of YC-1 to the sGC-CO complex alters the intensity pattern of RR bands assigned to the vinyl and propionate heme substituents, suggesting changes in the tilting of the pyrrole rings to which they are attached. YC-1 also shifts the RR intensity of the nu(FeC) and nu(CO) bands from 473 and 1985 cm(-1) to 487 and 1969 cm(-1), respectively, and induces an additional nu(FeC) band, at 521 cm(-1), assigned to five-coordinate heme-CO. Site-directed variants in the proximal heme pocket (P118A) or in the distal heme pocket (V5Y and I149Y) reduce the extent of YC-1 activation, along with the 473 cm(-1) band intensity. These lower-activity sGC variants display another nu(FeC) band at 493 cm(-1) which is insensitive to YC-1 addition and is attributed to protein that cannot be activated by the allosteric activator. The results are consistent with a model in which YC-1 binding to the sGC-CO complex results in a conformational change that activates the protein. Specifically, YC-1 binding alters the heme geometry via peripheral nonbonded contacts and also relieves an intrinsic electronic effect that weakens FeCO backbonding in the native, YC-1 responsive protein. This electronic effect might involve neutralization of the heme propionates via H-bond contacts or negative polarization by a distal cysteine residue. YC-1 binding also strains the Fe-histidine bond, leading to a population of the five-coordinate sGC-CO complex in addition to a conformationally distinct population of the six-coordinate sGC-CO complex. The loss of YC-1 activation in the sGC variants might involve a weakening of the heme-protein contacts that are thought to be critical to a YC-1-induced conformational change.
| S-EPMC2894327 | biostudies-literature
Project description:Nitric oxide (NO) signaling regulates key processes in cardiovascular physiology, specifically vasodilation, platelet aggregation, and leukocyte rolling. Soluble guanylate cyclase (sGC), the mammalian NO sensor, transduces an NO signal into the classical second messenger cyclic GMP (cGMP). NO binds to the ferrous (Fe(2+)) oxidation state of the sGC heme cofactor and stimulates formation of cGMP several hundred-fold. Oxidation of the sGC heme to the ferric (Fe(3+)) state desensitizes the enzyme to NO. The heme-oxidized state of sGC has emerged as a potential therapeutic target in the treatment of cardiovascular disease. Here, we investigate the molecular mechanism of NO desensitization and find that sGC undergoes a reductive nitrosylation reaction that is coupled to the S-nitrosation of sGC cysteines. We further characterize the kinetics of NO desensitization and find that heme-assisted nitrosothiol formation of ?1Cys-78 and ?1Cys-122 causes the NO desensitization of ferric sGC. Finally, we provide evidence that the mechanism of reductive nitrosylation is gated by a conformational change of the protein. These results yield insights into the function and dysfunction of sGC in cardiovascular disease.
| S-EPMC3522300 | biostudies-literature
Project description:Soluble guanylate cyclase (sGC) catalyses the synthesis of cyclic GMP in response to nitric oxide. The enzyme is a heterodimer of homologous α and β subunits, each of which is composed of multiple domains. We present here crystal structures of a heterodimer of the catalytic domains of the α and β subunits, as well as an inactive homodimer of β subunits. This first structure of a metazoan, heteromeric cyclase provides several observations. First, the structures resemble known structures of adenylate cyclases and other guanylate cyclases in overall fold and in the arrangement of conserved active-site residues, which are contributed by both subunits at the interface. Second, the subunit interaction surface is promiscuous, allowing both homodimeric and heteromeric association; the preference of the full-length enzyme for heterodimer formation must derive from the combined contribution of other interaction interfaces. Third, the heterodimeric structure is in an inactive conformation, but can be superposed onto an active conformation of adenylate cyclase by a structural transition involving a 26° rigid-body rotation of the α subunit. In the modelled active conformation, most active site residues in the subunit interface are precisely aligned with those of adenylate cyclase. Finally, the modelled active conformation also reveals a cavity related to the active site by pseudo-symmetry. The pseudosymmetric site lacks key active site residues, but may bind allosteric regulators in a manner analogous to the binding of forskolin to adenylate cyclase. This indicates the possibility of developing a new class of small-molecule modulators of guanylate cyclase activity targeting the catalytic domain.
| S-EPMC3591389 | biostudies-literature
Project description:Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low-level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to that of the xsNO state. In this study, the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains five-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents undergo changes in intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N-O stretching frequency increases, with no change in the Fe-NO stretching frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe-N-O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO.
| S-EPMC2883567 | biostudies-literature
Project description:Heme oxygenase-1 knockout, H(mox)1(-/-), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in H(mox)1(-/-) mice. The contractile responses [superior mesenteric arteries (SMA), from female H(mox)1(-/-) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in H(mox)1(-/-) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in H(mox)1(-/-) than H(mox)1(+/+) mice (p < 0.001). Branches from H(mox)1(-/-) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. H(mox)1(-/-) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were H(mox)1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of H(mox)1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of H(mox)1(-/-) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both α1 and β1 subunit levels were reduced to <50% of H(mox)1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of H(mox)1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.
| S-EPMC2957777 | biostudies-literature
Project description:Nitric oxide (NO) signaling mediates many important physiological processes through the receptor soluble guanylate cyclase (sGC). Under disease conditions sGC heme can be oxidized resulting in NO insensitivity. Here, we show that the therapeutic compound cinaciguat (Cin) rescues dysfunctional sGC by direct displacement of the oxidized heme.
| S-EPMC3477619 | biostudies-literature
Project description:In the nitric oxide (NO) signaling pathway, human soluble guanylate cyclase (hsGC) synthesizes cyclic guanosine monophosphate (cGMP); responsible for the regulation of cGMP-specific protein kinases (PKGs) and phosphodiesterases (PDEs). The crystal structure of the inactive hsGC cyclase dimer is known, but there is still a lack of information regarding the substrate-specific internal motions that are essential for the catalytic mechanism of the hsGC. In the current study, the hsGC cyclase heterodimer complexed with guanosine triphosphate (GTP) and cGMP was subjected to molecular dynamics simulations, to investigate the conformational dynamics that have functional implications on the catalytic activity of hsGC. Results revealed that in the GTP-bound complex of the hsGC heterodimer, helix 1 of subunit α (α:h1) moves slightly inwards and comes close to helix 4 of subunit β (β:h4). This conformational change brings loop 2 of subunit β (β:L2) closer to helix 2 of subunit α (α:h2). Likewise, loop 2 of subunit α (α:L2) comes closer to helix 2 of subunit β (β:h2). These structural events stabilize and lock GTP within the closed pocket for cyclization. In the cGMP-bound complex, α:L2 detaches from β:h2 and establishes interactions with β:L2, which results in the loss of global structure compactness. Furthermore, with the release of pyrophosphate, the interaction between α:h1 and β:L2 weakens, abolishing the tight packing of the binding pocket. This study discusses the conformational changes induced by the binding of GTP and cGMP to the hsGC catalytic domain, valuable in designing new therapeutic strategies for the treatment of cardiovascular diseases.
| S-EPMC7289801 | biostudies-literature
Project description:The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway plays a key role in the pathogenesis of pulmonary hypertension (PH). Targeted treatments include phosphodiesterase type 5 inhibitors (PDE5i) and sGC stimulators. The sGC stimulator riociguat is approved for the treatment of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). sGC stimulators have a dual mechanism of action, enhancing the sGC response to endogenous NO and directly stimulating sGC, independent of NO. This increase in cGMP production via a dual mechanism differs from PDE5i, which protects cGMP from degradation by PDE5, rather than increasing its production. sGC stimulators may therefore have the potential to increase cGMP levels under conditions of NO depletion that could limit the effectiveness of PDE5i. Such differences in mode of action between sGC stimulators and PDE5i could lead to differences in treatment efficacy between the classes. In addition to vascular effects, sGC stimulators have the potential to reduce inflammation, angiogenesis, fibrosis and right ventricular hypertrophy and remodelling. In this review we describe the evolution of treatments targeting the NO-sGC-cGMP pathway, with a focus on PH.
| S-EPMC10957071 | biostudies-literature
Project description:The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a β1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC "activators" aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules.
| S-EPMC8640258 | biostudies-literature