NO is generated

1. by nitric oxide synthase (NOS) 2. from NO2- (and NO3-)

1

Nitric Oxide Synthase (NOS)

Heme Fe(III)-S-Cys, Fused protein, H4B, Ca2+-Calmodulin, Home-Dimer, Zn2+-Cys n-NOS, e-NOS, i-NOS 2

NO3NO2-

3

Nature Chemical Biology 5, 865 (2009) Meeting Report

4

Major functions of NO are

-SNO formation Activation of soluble guanylate cyclase (sGC)

5

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6

Dual Role of Nitric Oxide Production in Cerebral Ischemic Injury

No more toxic SNO: signal transduction

7

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Figure 12. Summarized illustration of the main, significant categories of NO reactions in the vascular system. The reactions include (1) reactions with metal centers (mainly heme); (2) S-nitrosylation, or the interaction of NO with cysteine sulfahydryls/thiol, where a nitrosyl group is added post-translationally; (3) nitration (protein tyrosine); (4) free-radical interactions; (5) reactions with plasma O2; and (6) synthesis of cGMP through the catalysis of sGC by NO, then leading to the activation of protein kinases and phosphodiesterases.

8

Heme-based NO sensor protein Heme

Signal (NO)

sGC (soluble guanylate cyclase)

Sensor domain (Heme)

Functional domain

Protein structural change

Heme Sensor domain Functional domain Guanylate cyclase (NO-bound heme)

9

Important roles of Soluble guanylate cyclase (sGC) activated by NO Heme-based NO sensor pmol 200 times activation

10

Vasorelaxation Platelet Inhibition Soluble guanylate cyclase (sGC) sGC, eNOS: Heme protains

11

pmol NO 200 fold activation mmol CO 5 fold activation

12

13

Heme

Membrane-bound Hetero Dimer

Cyclic GMP 14

15

16

Figure 13. NO interactions with sGC and synthesis of cGMP. (A) Domain structure: sGC is a heterodimeric protein with the C-terminus serving as the catalytic domain and the N-terminus as the regulatory domain, which is sensitive to NO. (B) Structural organization: sGC is a protein present in the cytosol that is a hemeoprotein (a metalloprotein containing a heme prosthetic group) with subunits of α and β with a ferrous prosthetic group. NO interactions with sGC at a site that is alternative to heme (i.e., on a cysteine) are also recognized. The synthesis of cGMP through the catalysis of sGC with NO then leads to the activation of protein kinases and phosphodiesterases to modulate varied biochemical pathways that regulate vascular functions.(199) (C) Reactive site of sGC: Iron is ligated to histadine 105 of the β subunit; NO binds to the heme of sGC and forms a transient six-coordinated NO-bound state that progresses, upon heme–His bond breakage, to a five-coordinated NO-bound activated state. The degree of activation of sGC as high (where the FeNO bond angle is greater) and low (where the FeNO bond angle is smaller) could vary according to the amount of NO present, as well as other sGC stimulators. 17

Characterization of Two Different Five-Coordinate Soluble Guanylate Cyclase Ferrous–Nitrosyl Complexes Biochemistry 47, 3892 (2008).

18

19

A previously uncharacterized model for sGC activation and deactivation.

Cary S P L et al. PNAS 2005;102:13064-13069

20 ©2005 by National Academy of Sciences

Fig. 1.Models for NO activation of sGC. In the scheme depicted in black, NO binds rapidly to the basally active five-coordinate ferrous heme, forming a six-coordinate ferrous-nitrosyl intermediate. At a rate that depends on NO concentration, the final five-coordinate complex is activated several hundred-fold. In the scheme depicted in red, the modulation of the formation and dissociation of the sGC heme-NO complex is shown, as well as the activation state of ferrous-nitrosyl sGC, by ATP, GTP, and NO. 21

Model for activation of sGC. In the conversion of the 6C species to the 5C NO–heme complex (k3), NO acts as a catalyst in the reaction such that it is not consumed. Simulations (vide infra) are consistent with this. The step represented by k5 does not involve NO and shows NO bound on the distal and proximal sides of the heme. 22

soluble Guanylate Cyclase NO pmole 200-fold activated

23

Figure 14. NO-independent stimulators of soluble guanylate cyclase. Alternative sGC stimulators are currently under investigation in clinical trials. These novel compounds, which are still undergoing clinical trials, are promising, with the ability to resist oxidative stress-induced intolerance and desensitization of sGC. Some compounds are heme-dependent, namely, (a) YC-1, (b) BAY 41-2272, and (c) BAY63-2521 (riociguat), whereas others are heme-independent, namely, (d) BAY 58-2667 (cinaciguat) and (e) HMR1766 (ataciguat) and can act synergistically with NO to stimulate, activate, and prevent ubiqutin-mediated degradation.(214). 24

Figure 15. Summary of currently recognized factors for inducing and inhibiting NO, as well as factors directly influenced by NO. Pulsatile flow is a mechanical factor and arginine and BH4 are chemical factors that influence NOS and catalyze NO synthesis. NO donors and RBC can also induce NO synthesis, although RBC can also act as a scavenger of NO and inhibit NO-dependent activities. Oxidative stress acts to inhibit NO, NOS, and sGC, which is the main enzyme that NO activates to synthesize cGMP. sGC can be directly activated by heme-dependent/heme-independent activators, independent of NO, and is not influenced by the inhibitory effects of oxidative stress. 25

NOGC: soluble guanylyl cyclase, sGC 26

Table 1 | Main haem-dependent stimulators of soluble guanylate cyclase

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Table 2 | Main haem-independent activators of soluble guanylate cyclase

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Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein, Nitrophorin Proc. Natl. Acad. Sci. USA 102, 594 (2005)

Cimex lectularius (the bedbug)

Vasorelaxation + Platelet Inhibitio

Rhodnius prolixus (the kissing bug)

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Nitrophorin: NO carrier protein

30

31

Nitric Oxide-Induced Conformational Changes in Soluble Guanylate Cyclase Structure 22, 602 (2014) Soluble guanylate cyclase (sGC) is the primary mediator of nitric oxide (NO) signaling. NO binds the sGC heme cofactor stimulating synthesis of the second messenger cyclic-GMP (cGMP). As the central hub of NO/cGMP signaling pathways, sGC is important in diverse physiological processes such as vasodilation and neurotransmission. Nevertheless, the mechanisms underlying NO-induced cyclase activation in sGC remain unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to probe the NO-induced conformational changes of sGC. HDX-MS revealed NO-induced effects in several discrete regions. NO binding to the heme-NO/O2 -binding (H-NOX) domain perturbs a signaling surface implicated in Per/Arnt/Sim (PAS) domain interactions. Furthermore, NO elicits striking conformational changes in the junction between the PAS and helical domains that propagate as perturbations throughout the adjoining helices. Ultimately, NO binding stimulates the catalytic domain by contracting the active site pocket. Together, these conformational changes delineate an allosteric pathway linking NO binding to activation of the catalytic domain. 32

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Figure 6 Proposed NO-Induced Activation Mechanisms of sGC NO binding releases and opens the heme-associated helix of the H-NOX domain while condensing and closing the active site pocket of the catalytic domain. Dominant points of conformational articulation are highlighted in green. Higher-order domain architecture is based on an emerging single-particle EM study Campbell et al., 2014). The allosteric pathway bridging the sensor and output domains may involve two different mechanisms. (A) Large conformational changes at the junction between PAS and helical domains might indicate interdomain pivoting that relieves inhibitory contacts between H-NOX and catalytic domains. (B) Alternatively, the PAS-helical junction might mediate remote allosteric effects via long-range conformational changes propagated through the helical domains. 35

Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase Proc. Nat. Acad. Sci., USA 111, 2960 (2014)

Significance Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. Disruptions in NO signaling have been linked to hypertension, neurodegeneration, and heart disease. The mechanistic details underlying the modulation of sGC activity remain largely unknown. Determining the structure of full-length sGC is a prerequisite to understanding its function and for the design and improvement of therapeutics for treatment of related diseases. We use electron mnext-generation therapeuticsicroscopy to determine the quaternary structure of the protein. Furthermore, we found that both ligand-free and ligand-bound sGC are highly flexible. This structural information provides a significant step forward in understanding the mechanism of sGC activation and will ultimately empower the development of.

36

Abstract Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NOsignaling pathways mediate diverse physiological processes, including vasodilation, neurotransmission, and myocardial functions. sGC is a heterodimer assembled from two homologous subunits, each comprised of four domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length sGC. We used single-particle electron microscopy to obtain the structure of the complete sGC heterodimer and determine its higherorder domain architecture. Overall, the protein is formed of two rigid modules: the catalytic dimer and the clustered Per/Art/Sim and heme-NO/O2-binding domains, connected by a parallel coiled coil at two hinge points. The quaternary assembly demonstrates a very high degree of flexibility. We captured hundreds of individual conformational snapshots of free sGC, NO-bound sGC, and guanosine-5′- [(α,β)methylene]triphosphate-bound sGC. The molecular architecture and pronounced flexibility observed provides a significant step forward in understanding the mechanism of NO signaling.

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Fig. 5. Merged maps illustrating the range of motion available to sGC when free or ligand-bound. Maps were aligned to the catalytic domain and show a similar range of motion under several experimental conditions. (A) The sGC holoenzyme. (B) NObound sGC. (C) GPCPP-bound sGC. (D) GPCPP and NO-bound sGC. 41

42

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Therapeutic approaches targeting the nitric oxide (NO) signalling pathway in pulmonary hypertension. Riociguat stimulates soluble guanylate cyclase (sGC) with a dual mode of action. When sufficient NO is present, riociguat acts in synergy with NO, but it can also stimulate sGC directly when NO is absent or scarce. Phosphodiesterase (PDE)-5 inhibitors act further downstream in the pathway, preventing degradation of cyclic guanosine monophosphate (cGMP). Thus, their efficacy depends on the presence of an intact NO/sGC/cGMP signalling pathway. NOS: NO synthase; GMP: guanosine 45 monophosphate. Adapted from [26] with permission from the publisher.

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Proposed mechanism of high VT (HVT)-induced, cGMP-mediated endothelial barrier dysfunction. Solid arrows indicate either increased protein expression (where indicated) or activation. Dotted arrows indicate either decreased protein expression (where indicated) or inhibition. HVT ventilation increases eNOS/sGC-mediated cGMP production. At the same time, HVT increases PDE2A expression and decreases PDE3A expression. Under these circumstances, the effect of the increased cGMP from sGC is to activate PDE2A resulting in net loss of cAMP and worsened endothelial barrier function. L-arg, l-arginine; NO, nitric oxide.

47

Proposed pathways of H2S and NO interaction.

Coletta C et al. PNAS 2012;109:9161-9166

48 ©2012 by National Academy of Sciences

Heme-based gas sensor protein Heme Sensor domain Signal (O2, CO, NO) (Heme)

Functional domain

Protein structural change

Ec DOS, FixL, HemAT, sGC, CooA

Heme Sensor domain Functional domain Regulation of (Gas-bound heme)

catalysis and transcription 49

[A] histidine kinases - FixL (1), DosS and DosT (2), AfGcHK (3) and NtrY (5)

N

N

Fe N N

Histidine kinase

His ATP

SIGNAL

P

autophosphorylation

O2 dissociation (1,2) or association (3) or heme redox change (5)

response regulators - FixJ (1), DosR (2), NtrX (5)

Asp P phosphotransfer

OUPUT regulation of catalytic activity, transcription or chemotaxisis

Figure 4: Bacterial two-component system of [A] Fused type and [B] Stand-alone type. Physical stimuli, such as light illumination or oxygen association/dissociation, interact with the sensing site of a signal-sensing protein, initiating an autophosphorylation reaction at a histidine residue in the functional domain. The self-added phosphate group is ultimately transferred to an aspartate residue of the response regulator, triggering a functional response, such as DNA binding and initiation of transcription of an important protein.67,68 (1) FixL, a signal-sensing protein containing a hemebound PAS domain, adopts a heme Fe(II)-O2 complex form under resting conditions. Dissociation of O2 from the heme Fe(II) complex triggers the autophosphorylation reaction and subsequent phosphotransfer reaction to FixJ, a response regulator.41–43,66 (2) DosS and DosT are signaling proteins containing a heme-bound GAF domain.70–72,171 Their response regulator is DosR. Similar to FixL, O2 dissociation from the heme Fe(II) complex drives the autophosphorylation and phosphotransfer reaction to DosR. (3) AfGcHK is a signal-sensing protein containing a heme-bound globin domain.73 50 Unlike the case for FixL and DosS/DosT, O2 association with the heme Fe(II) complex significantly enhances autophosphorylation and phosphotransfer reactions to the response regulator.

Fig. 5. Schematic summary of the H-NOX family of heme-based sensors. The progenitor H-NOX domain has evolved to discriminate between ligands such as NO and O2 for specific sensing purposes. This is the first family of related heme proteins to 51 discriminate between different physiologically relevant diatomic gaseous ligands.

[B] sensor protein - HnoX (4) HnoX N

N

Fe N N

SIGNAL NO dissociation

HnoC, HnoD or HnoB response regulators

histidine kinase HnoK Histidine kinase

Asp

His ATP P autophosphorylation

P phosphotransfer

LuxU

HqsK Histidine kinase

Asp

ATP

P

His ATP

autophosphorylation

regulation of transcription or catalytic activity

LuxO

Histidine kinase

His P

OUPUT

P

Asp P

OUTPUT regulation of transcription

phosphotransfer Fig. 4

52

Fig. 4. The heme environment of the Tt H-NOX domain[38]. (a) The conserved Y-S-R motif makes hydrogen bonding interactions with the propionic acid side chains of the heme group, which is colored yellow (porphyrin) and red (iron). (b) The conserved H102 is the proximal ligand to the heme. In Tt H-NOX, a distal pocket hydrogen-bonding network, involving principally Y140, stabilizes an FeII–O2 complex. This hydrogen-bonding network is predicted to be absent in the H-NOX proteins from sGCs and aerobic prokaryotes, suggesting this as a key molecular factor in the remarkable ligand selectivity against O2 displayed by these heme proteins. 53