National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM128777
United States
National Institutes of Health/National Institute on Aging (NIH/NIA)
R01AG063845
United States
Citation
Journal: bioRxiv / Year: 2023 Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies. Authors: Natasha I Edman / Rachel L Redler / Ashish Phal / Thomas Schlichthaerle / Sanjay R Srivatsan / Ali Etemadi / Seong J An / Andrew Favor / Devon Ehnes / Zhe Li / Florian Praetorius / Max ...Authors: Natasha I Edman / Rachel L Redler / Ashish Phal / Thomas Schlichthaerle / Sanjay R Srivatsan / Ali Etemadi / Seong J An / Andrew Favor / Devon Ehnes / Zhe Li / Florian Praetorius / Max Gordon / Wei Yang / Brian Coventry / Derrick R Hicks / Longxing Cao / Neville Bethel / Piper Heine / Analisa Murray / Stacey Gerben / Lauren Carter / Marcos Miranda / Babak Negahdari / Sangwon Lee / Cole Trapnell / Lance Stewart / Damian C Ekiert / Joseph Schlessinger / Jay Shendure / Gira Bhabha / Hannele Ruohola-Baker / David Baker / Abstract: Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, ...Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways.
Evidence: gel filtration, SAXS, light scattering, multi-angle light scattering (MALS)
Type
Name
Symmetry operation
Number
identity operation
1_555
1
-
Components
#1: Protein
C8-71
Mass: 23435.004 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21 (bacteria)
-
Experimental details
-
Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
-
Sample preparation
Component
Name: Self-assembled homo-octamer of de novo designed protein C8-71 Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)
Organism: synthetic construct (others)
Source (recombinant)
Organism: Escherichia coli BL21 (bacteria)
Buffer solution
ID
Specimen-ID
pH
1
1
8
2
2
8
Specimen
Experiment-ID: 1 / Conc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample present within the ice layer as both isolated homo-octameric rings and fibrils of variable length
ID
1
2
Specimen support
ID
Specimen-ID
Grid material
Grid mesh size (divisions/in.)
Grid type
Details
1
1
COPPER
300
Quantifoil R2/2
2
2
COPPER
300
Quantifoil R1.2/1.3
No pre-treatment
Vitrification
Chamber temperature: 295 K / Cryogen name: ETHANE / Details: blot time = 4s; blot force = 0 / Entry-ID: 8F6Q / Humidity: 100 % / Instrument: FEI VITROBOT MARK IV
ID
Specimen-ID
1
1
2
2
-
Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Average exposure time: 2.5 sec. / Electron dose: 61.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3092
-
Processing
Software
Name
Version
Classification
NB
phenix.real_space_refine
1.16_3549
refinement
PHENIX
1.16_3549
refinement
EM software
ID
Name
Version
Category
Details
1
cryoSPARC
particleselection
2
Leginon
imageacquisition
4
cryoSPARC
CTFcorrection
CTFFIND
7
UCSF Chimera
modelfitting
9
cryoSPARC
initialEulerassignment
10
cryoSPARC
finalEulerassignment
12
cryoSPARC
3Dreconstruction
13
PHENIX
1.16
modelrefinement
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Symmetry
Point symmetry: C8 (8 fold cyclic)
3D reconstruction
Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132391 / Symmetry type: POINT
Atomic model building
Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficients Details: Designed oligomer was used as initial model and was initially fit into map using UCSF Chimera
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