Nitric Oxide-Induced Conformational Changes in Soluble Guanylate Cyclase

Underbakke, Eric S.; Iavarone, Anthony T.; Chalmers, Michael J.; Pascal, Bruce D.; Novick, Scott J.; Griffin, Patrick R.; Marletta, Michael A.

doi:10.1016/j.str.2014.01.008

Cited by 72 publications

(98 citation statements)

References 56 publications

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“…The functional importance of the PAS and helical domains in signal communication was previously revealed by mutational screens (25). In addition, the flexibility of the PAS-helical junction interestingly parallels the results of a recent HDX study that revealed NO-induced increases in conformational flexibility at the linker between the PAS and helical domains (14). Indeed, in several archetypal PAS domains, unfolding at the helical termini is thought to be important for signal communication (26,27).…”

Section: Discussionsupporting

confidence: 69%

“…H-NOX: Homology models of the sGC α1 and β1 H-NOX monomers were constructed from the structure of a bacterial H-NOX (PDB: 2O09) using Robetta (43) (14).…”

Section: Methodsmentioning

confidence: 99%

“…We have tentatively assigned the smaller lobe to the β1 H-NOX domain, which contains 66 fewer residues than its α1 counterpart. The α1 N-terminal extension, like many other targeting sequences, is thought to be largely unstructured (14). Thus, it would not provide a well-defined density in a single-particle EM map, making it difficult to unequivocally assign which H-NOX domain occupies which lobe.…”

Section: Figmentioning

confidence: 99%

“…1). The arrangement of and interactions between these domains have been further studied using a variety of techniques, including mutational and truncation studies, Förster resonance energy transfer (FRET), resonance Raman spectroscopy, chemical cross-linking, smallangle X-ray scattering (SAXS), and hydrogen deuterium exchange (HDX) (10)(11)(12)(13)(14). These studies have been used to propose a variety of models for the mechanisms of action of sGC, but the lack of a comprehensive 3D structure of the sGC holoenzyme has so far impeded a confident assignment of domain hierarchy.…”

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confidence: 99%

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Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase

Campbell

Underbakke

Potter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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/O 2 -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.N itric oxide (NO) has emerged as an integral signaling molecule in biology. Soluble guanylate cyclase (sGC), the primary receptor of NO in mammals, binds NO via an Fe II heme cofactor leading to a several hundred-fold increase in 3,5-cyclic guanosine monophosphate (cGMP) synthesis. cGMP then acts as a second messenger, targeting phosphodiesterases, ion-gated channels, and cGMP-dependent protein kinases. These target proteins go on to regulate many critical physiological functions including vasodilation, platelet aggregation, neurotransmission, and myocardial functions (1, 2). Disruptions in NO signaling have been linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (3, 4). sGC has been the focus of small-molecule modulators of activity for therapeutic advantage. Riociguat, which is a stimulator of sGC, has recently been approved for treatment of pulmonary hypertension (5). However, the mechanistic details underlying the modulation of sGC catalytic activity by NO and other small molecules remain largely unknown. Determining the structure of the full-length sGC, free and in complex with NO, is therefore a prerequisite to understanding its function and for the design and improvement of therapeutics for treatment of diseases involving the NO/cGMP pathway.The most extensively studied and physiologically relevant isoform of sGC is the 150-kDa heterodimer containing one α1 and one β1 subunit. Each subunit is comprised of four modular domains: the N-terminal heme-NO/O 2 -binding (H-NOX), the Per/Arnt/Sim (PAS), the helical, and the C-terminal catalytic domain (Fig. 1). No structure of the complete holoenzyme is available to date, and its absence precludes answering key questions such as how NO occupancy of the N-terminal β H-NOX sensor domain is communicated to the C-terminal cyclase domain. Atomic models of isola...

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Section: Discussionsupporting

confidence: 69%

“…H-NOX: Homology models of the sGC α1 and β1 H-NOX monomers were constructed from the structure of a bacterial H-NOX (PDB: 2O09) using Robetta (43) (14).…”

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase

Campbell

Underbakke

Potter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently, the entire quaternary structure of sGC was reconstructed by inserting individual protein domains into the density envelope of entire single-sGC molecules observed by EM (14), revealing a high flexibility of the sGC dimer. Subsequently, the structural perturbations induced by NO binding were mapped at the domain interfaces (15). In the past decade, a diversity of molecular models and regulatory models have been proposed (16)(17)(18)(19)(20)(21)(22)(23), with some including structural hypotheses and involving or not involving the hypothetical NO binding to the proximal heme side (vs. distal NO binding).…”

mentioning

confidence: 99%

Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

Yoo

Lamarre

Martin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τ G1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τ G2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10 8 M −1 ·s −1 . In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ 5C1 = 0.66 μs and τ 5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.soluble guanylate cyclase | nitric oxide | time-resolved absorption spectroscopy | allostery | protein activation T he soluble guanylate cyclase (sGC), localized in many different cell types, is the receptor of the endogenous messenger nitric oxide (NO) and catalyzes the formation of cGMP from GTP upon activation triggered by NO binding (1, 2). The diatomic messenger NO and sGC play a critical role in several physiological processes: regulation of vascular blood pressure and cardiovascular diseases (3), lung airway relaxation and pulmonary pathologies (4), immune response and inflammatory disorders (5), and tumor progression and apoptosis (6). Thus, sGC is a pharmacological target of very high interest, and several activators have been developed (7,8), leading to the approval of riociguat for the treatment of pulmonary hypertension (9, 10). Because of its pharmacological interest, the mechanisms of activation, deactivation, and regulation of sGC must be deciphered at the molecular level. Despite numerous efforts, the 3D crystal structure of heterodimeric sGC remains unknown, but the heme domain of the sGC β1-subunit [called heme NO/oxygen-binding (H-NOX)] was modeled from the heme domain of bacterial NO sensors (11,12) and the sGC catalytic α1-subunit was modeled from the catalytic α1-subunit of adenylate cyclase (13). Recently, the entire quaternary structure of sGC was reconstructed by inserting individual protein domains into the density envelope of entire single-sGC molecules observed by EM (14), revealing a high flexibility of the sGC dimer. Subsequently, the structural perturbations induced by NO bi...

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Nitrogen Monoxide (Nitric Oxide): Bioinorganic Chemistry

Fricker¹

2019

Encyclopedia of Inorganic and Bioinorganic Chemistry

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The discovery of nitrogen monoxide (nitric oxide, NO) as the vasodilator molecule endothelium‐derived relaxing factor revealed a major physiological role for this simple diatomic molecule. We now know that NO regulates cardiovascular function, is a neurotransmitter, and is a component of the innate immune response to infection and cancer. The physiological effects of NO are mediated by its varied chemistry which includes redox chemistry and interaction with transition metals. NO can interact with iron‐heme‐containing proteins including hemoglobin and cytochrome c oxidase, and nonheme iron proteins such as aconitase and bacterial transcription regulatory proteins. NO reacts with superoxide to form the highly reactive peroxynitrite which decomposes to give the hydroxyl radical. Nitrosothiols, thiol adducts of NO, act to control NO release, and to shuttle NO between proteins and transport NO across cell membranes. NO is produced by one of three nitric oxide synthase (NOS) enzymes, endothelial NOS, neuronal NOS, and inducible NOS. Signaling functions are mediated via interaction with the heme‐containing protein soluble guanylate cyclase (GC), which produces the cell signaling molecule cyclic guanosine monophosphate (cGMP). This, in turn, is hydrolyzed by phosphodiesterase 5 (PDE5), which turns off NO signaling. Perturbation in NO function contributes to the pathophysiology of many disease states. Endothelial dysfunction and subsequent reduced NO production contribute to hypertension and atherosclerosis. Excess NO is a mediator of inflammatory disease, and neurodegenerative disease, due in part to the production of peroxynitrite. The cardiovascular collapse observed in septic shock is caused by the vasodilator action of NO. Targeting the NO pathway theoretically provides many opportunities for therapeutic intervention. The NO donor drugs glyceryl trinitrate, isosorbide mononitrate, isosorbide dinitrate, and amyl nitrate are used to treat acute angina; and inhaled NO is used to treat persistent pulmonary hypertension in new born infants. Conversely, NOS inhibitors and NO scavengers have been explored to counteract NO as a mediator of disease. Though promising results have been obtained in animal models of disease these latter approaches have had little clinical success. However, targeting downstream of NO has been more successful with new clinically approved classes of drugs such as the inhibitors of PDE5 sildenafil, tadalafil, and vardenafil; and the stimulator of the NO receptor soluble GC, riociguat. It has become apparent that the biological actions, and biological chemistry, of NO are very complex. New opportunities for drug discovery could result from an improved understanding of the biology of NO.

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Nitric Oxide-Induced Conformational Changes in Soluble Guanylate Cyclase

Cited by 72 publications

References 56 publications

Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase

Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase

Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

Nitrogen Monoxide (Nitric Oxide): Bioinorganic Chemistry

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