1

NO is generated 1. by nitric oxide synthase (NOS) 2. from NO2- (and NO3-)

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

3

NO3-

NO2-

4 Nature Chemical Biology 5, 865 (2009) Meeting Report

5

Major functions of NO are

-SNO formation

Activation of soluble guanylate cyclase (sGC)

6

-----------------------

-------------------

Dual Role of Nitric Oxide Production in

Cerebral Ischemic Injury

7

No more toxic SNO: signal transduction

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

-----------------

9

Sensor domain

(Heme) Functional domain

Protein

structural

change

Sensor domain

(NO-bound heme)

Functional domain Guanylate cyclase

Signal (NO)

sGC (soluble guanylate cyclase)

Heme-based NO sensor protein

Heme

Heme

10

Important roles of

Soluble guanylate cyclase (sGC) activated by NO

Heme-based NO sensor

pmol 200 times activation

11

Vasorelaxation

Platelet Inhibition

Soluble guanylate

cyclase (sGC)

sGC, eNOS: Heme protains

12

pmol NO 200 fold activation mmol CO 5 fold activation

13

Cyclic GMP

Membrane-bound Hetero Dimer

Heme

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

©2005 by National Academy of Sciences

20

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

23

soluble Guanylate Cyclase

NO pmole

200-fold activated

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

27

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

28

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

Heme-assisted S-nitrosation of a proximal thiolate in

a nitric oxide transport protein, Nitrophorin

Proc. Natl. Acad. Sci. USA 102, 594 (2005)

29 Rhodnius prolixus (the kissing bug)

Cimex lectularius (the bedbug)

Vasorelaxation + Platelet Inhibition

30 Nitrophorin: NO carrier protein

31

32

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.

33

34

35

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.

36

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.

37

Abstract

Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in

mammals and a central component of the NO-signaling pathway. The NO-

signaling 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 higher-

order 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.

38

39

40

41

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) NO-

bound sGC. (C) GPCPP-bound sGC. (D) GPCPP and NO-bound

sGC.

42

43

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 monophosphate. Adapted from [26] with permission from the publisher.

45

46

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

©2012 by National Academy of Sciences

48

49

Sensor domain

(Heme) Functional domain

Protein

structural

change

Sensor domain

(Gas-bound heme)

Functional domain Regulation of

catalysis and

transcription

Signal (O2, CO, NO)

Ec DOS, FixL, HemAT, sGC, CooA

Heme-based gas sensor protein

Heme

Heme

FeN N

NN Histidine kinase

His P

Asp

P

histidine kinases - FixL (1), DosS and

DosT (2), AfGcHK (3) and NtrY (5)

response regulators - FixJ (1),

DosR (2), NtrX (5)

ATP

SIGNAL

O2 dissociation (1,2) or association

(3) or heme redox change (5)

autophosphorylation

phosphotransfer

OUPUT regulation of

catalytic activity,

transcription or

chemotaxisis

[A]

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 heme-bound 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 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.

50

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

discriminate between different physiologically relevant diatomic gaseous ligands. sGC and the aerobic prokaryotic H-NOX proteins bind NO in the presence of orders

51

Fig. 4

[B] sensor protein - HnoX (4)

FeN N

NN

Histidine kinase

His P

Asp

P

HnoC, HnoD or HnoB

response regulators histidine kinase

ATP

autophosphorylation phosphotransfer

OUPUT

regulation of

transcription or

catalytic activity

SIGNAL

NO dissociation

Histidine kinase

His P

HqsK

ATP

Asp

P

Histidine kinase

His

LuxU

ATP P

Asp

P OUTPUT

regulation of

transcription

LuxO

HnoX

HnoK

autophosphorylation phosphotransfer

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