Protein compactness and interaction valency define the architecture of a biomolecular condensate across scales
- Max Perutz Labs, Vienna Biocenter Campus (VBC), Austria
- University of Vienna, Center for Molecular Biology, Department of Structural and Computational Biology, Austria
- Medical University of Vienna, Center for Medical Biochemistry, Austria
- MM Shemyakin and Yu A Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russian Federation
- University of Vienna, Center for Molecular Biology, Department of Biochemistry and Cell Biology, Austria
Figures
Figure 1 with 2 supplements
Lge11-80 condensate formation critically depends on tyrosine residues.
(A) Sequence of Lge11-80. Arginines and tyrosines are highlighted in deep blue and magenta, respectively. (B) Condensate formation for Lge11-80 WT (left) and R>K (middle) in buffer with 200 mM NaCl. …
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Figure 1—source data 1
Raw data used in panel C.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig1-data1-v2.zip
Figure 1—figure supplement 1
Experimental and modeling studies of the effect of tyrosine and arginine mutations in Lge11-80.
(A) Lge11-80 purification and purity. Following purification, the constructs were analyzed by SDS-PAGE (4–12% gel, MOPS buffer) and Coomassie staining. Lge11-80 purity was assessed by densitometry, …
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Figure 1—figure supplement 1—source data 1
Raw data used in panels A and C.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Calculated free-energy values used in panel E.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig1-figsupp1-data2-v2.zip
Figure 1—figure supplement 2
Fluorescence recovery after photobleaching (FRAP) analyses of Lge11-80 and LAF-1 condensates.
FRAP curve of Dylight-labeled Lge11-80 WT (orange) and R>K (gray) condensates that were bleached in the center (A), the periphery (B), or across the whole condensate (C). Recovery of the normalized …
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Figure 1—figure supplement 2—source data 1
Fluorescence recovery after photobleaching (FRAP) summary: fitting parameters, recovery half-times, and raw data.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig1-figsupp2-data1-v2.xlsx
Figure 2 with 1 supplement
Analysis of interaction networks for Lge11-80 variants by all-atom molecular dynamics (MD) in 24-copy systems.
(A) Exemplary MD snapshot of the WT interaction network (see Video 3 for the full MD trajectory). Proteins in the simulation box are given in the atomic representation (orange), whereby glycine, …
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Figure 2—source data 1
Contact statistics used in panel B.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig2-data1-v2.xlsx
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Figure 2—source data 2
Molecular dynamics (MD) data used in panels C, D, E.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig2-data2-v2.zip
Figure 2—figure supplement 1
Inter- and intramolecular interactions of Lge11-80 variants in molecular dynamics (MD) simulations.
(A) Top 10 most frequent and enriched pairwise contacts between the residues in different protein molecules in multi-chain simulations (percentages, % 24; enrichment, Enr 24) as compared to …
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Figure 2—figure supplement 1—source data 1
Molecular dynamics (MD) data used in panels A, B, C.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig2-figsupp1-data1-v2.zip
Figure 3 with 1 supplement
Lge11-80 variants exhibit a dynamic binding mode in multi-chain systems.
(A) Representative distributions of statistically defined interaction regions (‘stickers’) mapped onto the protein sequence. Protein sequences are colored according to the average contact statistics …
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Figure 3—source data 1
Contact statistics used in panel A.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig3-data1-v2.zip
Figure 3—figure supplement 1
Correspondence between inter- and intramolecular interaction modes of Lge11-80 variants.
(A) Distributions of statistically defined interaction regions (‘stickers’) along the protein sequence in the single-chain systems. Protein sequences are colored according to the average contact …
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Figure 3—figure supplement 1—source data 1
Contact statistics used in panel A and correlation data shown in panels B and C.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig3-figsupp1-data1-v2.zip
Figure 4 with 1 supplement
Impact of Lge11-80 sequence on its conformational behavior and dynamics.
Distributions of radii of gyration (Rg) for Lge11-80 variants in (A) single-chain and (B) multi-chain systems. The last 0.3 µs of molecular dynamics (MD) trajectories were used to collect Rg …
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Figure 4—source data 1
Molecular dynamics (MD) data used in panels A, B, D, E, and F.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig4-data1-v2.zip
Figure 4—figure supplement 1
Conformational behavior and condensate rheology of Lge11-80 variants.
(A) Molecular dynamics (MD) conformations of WT (top panels), Y>A (middle panels), and R>K (bottom panels) variants illustrating different levels of compactness with the corresponding inter-residue …
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Figure 4—figure supplement 1—source data 1
Molecular dynamics (MD) data used in panels B, C, D, and E.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig4-figsupp1-data1-v2.zip
Figure 5 with 2 supplements
Describing condensate architecture via a fractal scaling model.
(A) Schematic representation of a scaling principle in condensate assembly. Interaction valency (n) and compactness (φ) of individual proteins determine the properties of protein clusters at …
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Figure 5—source data 1
Numerical data used in panel B.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig5-data1-v2.zip
Figure 5—figure supplement 1
Protein clusters and condensate topology of Lge11-80.
(A) A 24-protein cluster for Lge11-80 WT fitted to an ellipsoid with radii corresponding to x, y, z components of Rg. Protein atoms are shown as spheres. (B) Power-law relationship between mass and …
Figure 5—figure supplement 2
Parameters used in the fractal model for different Lge11-80 variants (related to Figures 5 and 6).
Radius of gyration (Rg), valency (n), and compactness (φ) averaged over the last 0.3 µs of molecular dynamics (MD) trajectories for all 24 copies. Slope (A) and intercept (B) of the linear …
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Figure 5—figure supplement 2—source data 1
Numerical data shown in the figure (fractal model parameters).
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig5-figsupp2-data1-v2.xlsx
Figure 6 with 1 supplement
Reconstruction of the large-scale condensate architecture with atomistic resolution.
(A) Transformation of a coarse-grained 1024 particle cluster obtained by FracVAL algorithm to an all-atom representation. The cluster was reconstructed using the fractal dimension df and the …
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Figure 6—source data 1
Numerical data used in panel C.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig6-data1-v2.zip
Figure 6—figure supplement 1
Partitioning of dextran of different sizes into condensates formed by Lge11-80 WT.
(A) Lge11-80 condensates are permeable to dextran of different sizes (partition ratio ≥1). Mean and st. dev. are indicated. n=100 condensates. (B) Condensates were incubated with TRITC-labeled …
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Figure 6—figure supplement 1—source data 1
Dextran partitioning raw data used in panels A and B.
- https://cdn.elifesciences.org/articles/80038/elife-80038-fig6-figsupp1-data1-v2.zip
Videos
Video 1
Fusion of Lge11-80 WT condensates in solution.
Protein concentration 1 µM. Scale bar, 5 µm.
Video 2
Fusion of Lge11-80 R>K condensates in solution.
Protein concentration 10 µM. Scale bar, 5 µm.
Video 3
Lge11-80 polypeptides self-associate in the crowed environment.
All-atom, explicit-solvent molecular dynamics (MD) simulation with 24 copies, corresponding to the concentration of ~7 mM, of WT Lge11-80. The movie shows the complete 1 µs of the simulated …
Video 4
Zoom-in of the internal organization of the WT Lge11-80 condensate at all-atom resolution.
1024-particle cluster was obtained by the FracVAL algorithm and transformed to an all-atom representation (see Methods). Protein atoms are shown as spheres. Video shows ×10 magnification.
Video 5
Zoom-in of the internal organization of the R>K Lge11-80 condensate at all-atom resolution.
1024particle cluster was obtained by the FracVAL algorithm and transformed to an all-atom representation (see Methods). Protein atoms are shown as spheres. Video shows ×15 magnification.
Video 6
Zoom-in of the internal organization of the Y>A Lge11-80 condensate at all-atom resolution.
1024-particle cluster was obtained by the FracVAL algorithm and transformed to an all-atom representation (see Methods). Protein atoms are shown as spheres. Video shows ×30 magnification.
Tables
Table 1
Details of simulated systems including composition, effective molar and mass protein concentration, size of the simulated cubic box, simulation time, and the number of replicas.
| Name | Protein | Water | Na+ | Cl- | [Protein], mM | [Protein], g/l | Box size, nm | MD time, µs | Replicas |
|---|---|---|---|---|---|---|---|---|---|
| Lge1 1–80 WT | 1 | 23674 | 44 | 50 | 2.3 | 20.7 | 9.0 | 1 | 2 |
| Lge1 1–80 Y>A | 1 | 23742 | 44 | 50 | 2.3 | 17.7 | 9.0 | 1 | 2 |
| Lge1 1–80 R>K | 1 | 23673 | 44 | 50 | 2.3 | 20.0 | 9.0 | 1 | 2 |
| Lge1 1–80 WT 24 copies | 24 | 182056 | 351 | 495 | 6.9 | 62.5 | 18.0 | 1 | 1 |
| Lge1 1–80 Y>A 24 copies | 24 | 183407 | 351 | 495 | 6.9 | 45.0 | 18.0 | 1 | 1 |
| Lge1 1–80 R>K 24 copies | 24 | 217857 | 413 | 557 | 5.8 | 50.8 | 19.0 | 1 | 1 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Saccharomyces cerevisiae) | LGE1 | SGD databank | YPL055C | Mutants used in this work were described in Gallego et al., 2020 |
| Strain, strain background (Escherichia coli) | BL21 CodonPlus (DE3)-RIL | Stratagene | #200131 | Chemically competent cells |
| Other | (TRITC)-labeled dextran, Mw 155 KDa | Sigma-Aldrich | #T1287 | Final concentration 0.05 mg/ml |
| Other | (TRITC)-labeled dextran, Mw 65–85 KDa | Sigma-Aldrich | #T1162 | Final concentration 0.05 mg/ml |
| Other | (TRITC)-labeled dextran, Mw 2000 KDa | Thermo Fisher | #D7139 | Final concentration 0.05 mg/ml |
| Other | Dylight 488 NHS-Ester | Thermo Fisher | #46402 | Methods in this paper |
| Software, algorithm | ImageJ 1.53t | https://imagej.nih.gov/ij | Version 1.53t | |
| Software, algorithm | GraphPad Prism 7.0e | https://www.graphpad.com | Version 7.0e |
Additional files
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Supplementary file 1
Technical summary on molecular dynamics (MD) results.
- https://cdn.elifesciences.org/articles/80038/elife-80038-supp1-v2.xlsx
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Supplementary file 2
Diffusion summary.
- https://cdn.elifesciences.org/articles/80038/elife-80038-supp2-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/80038/elife-80038-mdarchecklist1-v2.pdf
