Molecular Model of a Soluble Guanylyl Cyclase Fragment Determined by Small-Angle X-ray Scattering and Chemical Cross-Linking

Fritz, Bradley G.; Roberts, S.A.; Ahmed, Aqeel; Breci, Linda; Li, Wenzhou; Weichsel, A.; Brailey, Jacqueline L.; Wysocki, Vicki H.; Tama, Florence; Montfort, W.R.

doi:10.1021/bi301570m

Cited by 54 publications

(104 citation statements)

References 70 publications

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“…We clearly establish here that the PAS and H-NOX domains directly interact, as previously suggested (12,13). Moreover, we show that these two domains form a tight cluster, sharing large surfaces of interactions, and allow each H-NOX domain to interact with both the α1 and β1 PAS domain.…”

Section: Discussionsupporting

confidence: 87%

“…Our assignment is similar to a proposed model based primarily on truncation, crosslinking, and SAXS data (12). Due to the crossing of the helical domains in the coiled-coil arrangement, the β1 H-NOX domain is poised to interact with the α1 subunit of the catalytic dimer, consistent with mounting structural and biochemical data (11)(12)(13). The EM maps indicate asymmetry in the elongated lobes: one is slightly smaller than the other.…”

Section: Figsupporting

confidence: 85%

“…The remaining H-NOX and PAS domains fill the four lobes of the larger, 90 × 35 × 45 Å density. The overall 3D shape of this region of density closely resembles the previously reported SAXS envelope obtained for a truncated construct of Manduca sexta sGC that did not include the catalytic domain (12). The volume of the corresponding EM density is slightly smaller than the SAXS envelope (115 × 70 × 90 Å), likely due to the presence of the catalytic domain that prevents the helical domain from folding over on the PAS/H-NOX cluster.…”

Section: Figsupporting

confidence: 79%

“…The structure of the rat sGC β1 monomer helical domain was previously determined and is formed by a long 13-turn helix followed by a shorter 2.5-turn helix (8). Although crystallized as an antiparallel homodimer, its arrangement in native sGC is considered to be a parallel heterodimer (8,12,20). The thin stalk of density observed in our 3D reconstructions accounts for this coiled coil, and a parallel arrangement of the helices is confirmed as it is the only Fig.…”

Section: Helical Coiled-coil Domain Forms the Elongated Stalk And Promentioning

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: 87%

Section: Figsupporting

confidence: 85%

Section: Figsupporting

confidence: 79%

Section: Helical Coiled-coil Domain Forms the Elongated Stalk And Promentioning

confidence: 99%

mentioning

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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Inhibition of soluble guanylyl cyclase by small molecules targeting the catalytic domain

et al. 2016

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Soluble guanylyl cyclase (sGC) plays a crucial role in cyclic nucleotide signaling that regulates numerous important physiological processes. To identify new sGC inhibitors that may prevent the formation of the active catalytic domain conformation, we carried out an in silico docking screen targeting a ‘backside pocket’ of the inactive sGC catalytic domain structure. Compounds 1 and 2 were discovered to inhibit sGC even at high/saturating nitric oxide concentrations. Both compounds also inhibit the BAY 58-2667-activated sGC as well as BAY 41-2272-stimulated sGC activity. Additional biochemical analyses showed that compound 2 also inhibits the isolated catalytic domain, thus demonstrating functional binding to this domain. Both compounds have micromolar affinity for sGC and are potential leads to develop more potent sGC inhibitors.

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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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Molecular Model of a Soluble Guanylyl Cyclase Fragment Determined by Small-Angle X-ray Scattering and Chemical Cross-Linking

Cited by 54 publications

References 70 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

Inhibition of soluble guanylyl cyclase by small molecules targeting the catalytic domain

Nitrogen Monoxide (Nitric Oxide): Bioinorganic Chemistry

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