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- PDB-9csd: The Ectodomains of SPRING and S1P with the inhibitor PF-429242 -

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Basic information

Entry
Database: PDB / ID: 9csd
TitleThe Ectodomains of SPRING and S1P with the inhibitor PF-429242
Components
  • Membrane-bound transcription factor site-1 protease
  • SREBP regulating gene protein
KeywordsMEMBRANE PROTEIN/INHIBITOR / protease / complex / inhibitor / MEMBRANE PROTEIN / MEMBRANE PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


site-1 protease / CREB3 factors activate genes / positive regulation of SREBP signaling pathway / ATF6-mediated unfolded protein response / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / regulation of cholesterol biosynthetic process / Assembly of active LPL and LIPC lipase complexes / membrane protein intracellular domain proteolysis / regulation of vesicle-mediated transport ...site-1 protease / CREB3 factors activate genes / positive regulation of SREBP signaling pathway / ATF6-mediated unfolded protein response / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / regulation of cholesterol biosynthetic process / Assembly of active LPL and LIPC lipase complexes / membrane protein intracellular domain proteolysis / regulation of vesicle-mediated transport / Regulation of cholesterol biosynthesis by SREBP (SREBF) / Golgi stack / lysosome organization / mitotic G2 DNA damage checkpoint signaling / endoplasmic reticulum unfolded protein response / cholesterol metabolic process / protein maturation / response to endoplasmic reticulum stress / Post-translational protein phosphorylation / protein processing / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / protein import into nucleus / endoplasmic reticulum lumen / Golgi membrane / serine-type endopeptidase activity / endoplasmic reticulum membrane / Golgi apparatus / proteolysis
Similarity search - Function
SREBP regulating gene protein / SREBP regulating gene protein / Site-1 peptidase catalytic domain / : / Membrane-bound Site-1 Protease Family N-terminal domain / MBTPS1, fourth GATase-like domain / MBTPS1, third Ig-like domain / : / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. ...SREBP regulating gene protein / SREBP regulating gene protein / Site-1 peptidase catalytic domain / : / Membrane-bound Site-1 Protease Family N-terminal domain / MBTPS1, fourth GATase-like domain / MBTPS1, third Ig-like domain / : / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
: / Membrane-bound transcription factor site-1 protease / SREBP regulating gene protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKober, D.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM155152 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis for substrate selectivity by site-one protease revealed by studies with a small-molecule inhibitor.
Authors: Ashley V Bullington / Ilaria Micallo / Bilkish Bajaj / Pankaj Kumar / Netanya Schlamowitz / Aurora Silva / Sebastian Hendrix / Noam Zelcer / Daniel L Kober /
Abstract: Site-one protease (S1P) carries out the first proteolytic step to activate membrane-bound effector proteins in the Golgi. S1P matures through an autocatalytic process that begins in the endoplasmic ...Site-one protease (S1P) carries out the first proteolytic step to activate membrane-bound effector proteins in the Golgi. S1P matures through an autocatalytic process that begins in the endoplasmic reticulum (ER) and culminates with the displacement of its inhibitory pro-domain by its cofactor, sterol regulatory element binding protein-regulating gene (SPRING). Spatial control of S1P activity and substrate localization underpins signaling pathways governing, among others, lipogenesis, ER stress, and lysosome biogenesis. The factors governing these pathways are activated by S1P-mediated proteolysis upon their regulated transport from the ER to the Golgi. S1P cleaves substrates with the recognition sequence RX(L/I/V)Z, where X is any residue other than Cys or Pro and Z is preferably Leu or Lys. However, the structural basis for substrate recognition by S1P has remained unknown. Here, we used the small molecule PF-429242, a competitive inhibitor of S1P, to investigate substrate recognition by the S1P/SPRING complex. We determined the structure of S1P/SPRING bound to PF-429242 and found that PF-429242 binds S1P in the same pocket that recognizes the substrate's conserved P Arg. Further structural analysis suggests that S1P requires a conformation change to accommodate the substrate's P (L/I/V) residue. We designed an S1P mutation (I308A) to reduce the steric clash at the P position and generated an S1P that was resistant to PF-429242 in biochemical and cell culture assays. Our findings reveal selectivity in the recognition of substrates by S1P and provide a roadmap for the rational design of improved S1P inhibitors.
History
DepositionJul 23, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Membrane-bound transcription factor site-1 protease
B: SREBP regulating gene protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,5066
Polymers138,2482
Non-polymers1,2574
Water75742
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Membrane-bound transcription factor site-1 protease


Mass: 115048.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Construct contains 1-998 of human site one protease followed by a 3xFLAG affinity tag
Source: (gene. exp.) Homo sapiens (human) / Gene: MBTPS1 / Production host: Homo sapiens (human) / References: UniProt: Q14703
#2: Protein SREBP regulating gene protein / SREBF pathway regulator in Golgi 1


Mass: 23200.291 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Construct contains an IgK leader peptide followed by human SPRING residues 35-205 followed by a 10-His tag
Source: (gene. exp.) Homo sapiens (human) / Gene: SPRING1, C12orf49, SPRING / Production host: Homo sapiens (human) / References: UniProt: Q9H741

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Sugars , 2 types, 2 molecules

#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 44 molecules

#5: Chemical ChemComp-A1AZU / 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-[(3R)-pyrrolidin-3-yl]benzamide


Mass: 409.564 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H35N3O2 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Ca
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 42 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: Complex of the ectodomains of S1P and SPRING in the presence of the small molecule PF-429242
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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: 2000 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 64 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
10cryoSPARC4.4final Euler assignment
11cryoSPARC4.4classification
12cryoSPARC4.43D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122723 / Symmetry type: POINT
RefinementHighest resolution: 2.4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046845
ELECTRON MICROSCOPYf_angle_d0.5479312
ELECTRON MICROSCOPYf_dihedral_angle_d5.3361010
ELECTRON MICROSCOPYf_chiral_restr0.0441005
ELECTRON MICROSCOPYf_plane_restr0.0041206

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