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- PDB-9oxn: Compact, ligand-free state of Manduca sexta soluble guanylate cyc... -

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Basic information

Entry
Database: PDB / ID: 9oxn
TitleCompact, ligand-free state of Manduca sexta soluble guanylate cyclase mutant beta C122S
Components
  • Guanylate cyclase soluble subunit beta-1
  • Soluble guanylyl cyclase alpha-1 subunit
KeywordsSIGNALING PROTEIN / Cyclase / NO
Function / homology
Function and homology information


guanylate cyclase complex, soluble / guanylate cyclase / guanylate cyclase activity / response to oxygen levels / : / heme binding / GTP binding
Similarity search - Function
Haem NO binding associated / Haem NO binding associated domain superfamily / Heme NO binding associated / Heme NO-binding / H-NOX domain superfamily / Haem-NO-binding / Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / NO signalling/Golgi transport ligand-binding domain superfamily / Adenylyl- / guanylyl cyclase, catalytic domain ...Haem NO binding associated / Haem NO binding associated domain superfamily / Heme NO binding associated / Heme NO-binding / H-NOX domain superfamily / Haem-NO-binding / Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / NO signalling/Golgi transport ligand-binding domain superfamily / Adenylyl- / guanylyl cyclase, catalytic domain / Adenylate and Guanylate cyclase catalytic domain / Adenylyl cyclase class-3/4/guanylyl cyclase / Guanylate cyclase domain profile. / Nucleotide cyclase
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / guanylate cyclase / Guanylate cyclase soluble subunit beta-1
Similarity search - Component
Biological speciesManduca sexta (tobacco hornworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsThomas, W.C. / Houghton, K.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5F32GM149060-02 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24-GM129541-02 United States
CitationJournal: Biochemistry / Year: 2025
Title: Molecular Aspects of Soluble Guanylate Cyclase Activation and Stimulator Function.
Authors: Kimberly A Houghton / William C Thomas / Michael A Marletta /
Abstract: Soluble guanylate cyclases (sGCs) are heme-containing, gas-sensing proteins which catalyze the formation of cGMP from GTP. In humans, sGCs are highly selective sensors of nitric oxide (NO) and play a ...Soluble guanylate cyclases (sGCs) are heme-containing, gas-sensing proteins which catalyze the formation of cGMP from GTP. In humans, sGCs are highly selective sensors of nitric oxide (NO) and play a critical role in NO-based regulation of cardiovascular and pulmonary function. The physiological importance of sGC signaling has led to the development of drugs, known as stimulators and activators, which increase sGC catalytic function. Here we characterize a newly developed stimulator, CYR715, which is a particularly potent stimulator of () sGC catalytic function even in the absence of NO, increasing activity of the NO-free enzyme to 45% of full catalytic activity. CYR715 also increased the catalytic activity of sGC βC122A and βC122S variants, with a marked stimulation of the NO-free βC122S variant to 74% of maximum. High-resolution cryo-electron microscopy structures were solved for CYR715 bound to sGC βC122S revealing that CYR715 occupies the same binding site as the characterized sGC stimulators YC-1 and riociguat. Additionally, the core scaffold of CYR715 makes a binding interaction with βC78 while the flexible tail can interact with αR429 or βY7 and E361. Conformational extension of sGC following NO, YC-1, or CYR715 binding was characterized using small-angle X-ray scattering, revealing that while ligand binding results in sGC extension this extension does not directly correlate to observed activity. This suggests that not all conformational extensions of sGC result in increased catalytic activity, and that effective stimulators assist in converting extension into catalytic function.
History
DepositionJun 3, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Soluble guanylyl cyclase alpha-1 subunit
B: Guanylate cyclase soluble subunit beta-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,3813
Polymers146,7652
Non-polymers6161
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Soluble guanylyl cyclase alpha-1 subunit


Mass: 78598.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Manduca sexta (tobacco hornworm)
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
References: UniProt: O77105
#2: Protein Guanylate cyclase soluble subunit beta-1 / Guanylate cyclase soluble subunit beta-3 / Soluble guanylate cyclase small subunit


Mass: 68165.930 Da / Num. of mol.: 1 / Mutation: C122S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Manduca sexta (tobacco hornworm)
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
References: UniProt: O77106, guanylate cyclase
#3: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ligand-free Manduca sexta soluble guanylase cyclase variant
Type: COMPLEX
Details: Heterodimeric sGC molecule in the ligand-free, compact state. Beta-C122S mutant variant.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: .147 MDa / Experimental value: NO
Source (natural)Organism: Manduca sexta (tobacco hornworm)
Source (recombinant)Organism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTriethanolamine (TEA)C6H15NO31
225 mMSodium chlorideNaCl1
35 mMDithiothreitol (DTT)C4H10O2S21
45 mMMagnesium chlorideMgCl21
50.5 mMFluorinated Octyl MaltosideC20H25F13O111
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Samples were prepared in a Coy anaerobic chamber at RT. Protein was thawed at 4 C, reduced with 10 mM Na2S2O4 for 15 minutes at RT, and desalted using a Zeba spin column equilibrated with ...Details: Samples were prepared in a Coy anaerobic chamber at RT. Protein was thawed at 4 C, reduced with 10 mM Na2S2O4 for 15 minutes at RT, and desalted using a Zeba spin column equilibrated with Buffer, 0.22 um filtered. Protein samples were then diluted to 10 uM in equivalent buffer but with addition of 0.5 mM FOM.
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Cryo-EM samples were prepared by applying 3 ul to a glow-discharged Quantifoil R1.2/1.3 holey-carbon cryo-EM grid. The grid was blotted for 4 s with Whatman #1 filter paper and then plunge- ...Details: Cryo-EM samples were prepared by applying 3 ul to a glow-discharged Quantifoil R1.2/1.3 holey-carbon cryo-EM grid. The grid was blotted for 4 s with Whatman #1 filter paper and then plunge-frozen in liquid ethane with a Mark IV Vitrobot (ThermoFisher) at 4 C and 100% humidity.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.2195 sec. / Electron dose: 1.25 e/Å2 / Film or detector model: FEI FALCON I (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11872

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIX1.21.1_5286model refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1226641
Details: Autopicking was used to produce a stack of 1,226,641 particles. Particles showing poor alignment or broken complexes were removed using a series of 2D classification steps, leaving a ...Details: Autopicking was used to produce a stack of 1,226,641 particles. Particles showing poor alignment or broken complexes were removed using a series of 2D classification steps, leaving a particle stack of 368,563 particles that resembled sGC dimers.
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264227
Details: During refinement it became clear that there was minor interdomain flexibility within the 264,227 particles. In particular, the catalytic domain was at lower resolution and had considerable ...Details: During refinement it became clear that there was minor interdomain flexibility within the 264,227 particles. In particular, the catalytic domain was at lower resolution and had considerable missing density. Separate masks were created for each domain, and local refinement as implemented in cryoSPARC was used to improve the resolution of the separate catalytic and H-NOX domains of the global map. The subsequent local refinement resulted in 3.0 A and 3.4 A maps for the H-NOX and catalytic domains respectively. Both have improved density for outer regions of the map and improved local resolution. A composite map was used for model building.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Refinement was performed using iterative rounds of Phenix real space refinement and manual modeling in Coot. Phenix refinement was performed for separate domains of the model using the ...Details: Refinement was performed using iterative rounds of Phenix real space refinement and manual modeling in Coot. Phenix refinement was performed for separate domains of the model using the higher-resolution local maps of those domains.
Atomic model buildingDetails: ModelAngelo and Alphafold / Source name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029298
ELECTRON MICROSCOPYf_angle_d0.4912620
ELECTRON MICROSCOPYf_dihedral_angle_d8.4691269
ELECTRON MICROSCOPYf_chiral_restr0.0391463
ELECTRON MICROSCOPYf_plane_restr0.0061596

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