Multivalency regulates activity in an intrinsically disordered transcription factor
- Sarah Clark
- Janette B Myers
- Ashleigh King
- Radovan Fiala
- Jiri Novacek
- Grant Pearce
- Jörg Heierhorst
- Elisar J Barbar
- Oregon State University, United States
- Portland State University, United States
- The University of Melbourne, Australia
- Masaryk University, Czech Republic
- University of Canterbury, New Zealand
Figures
Figure 1
LC8 dimerizes its protein partners
(a) Ribbon diagram of dimeric LC8, where each monomer is colored a different shade of blue and bound to a representative peptide in yellow (PDB 5E0L). A single LC8 dimer binds two peptides …
-
Figure 1—source data 1
A list of the 10-amino acid motifs and PDB identification codes for the 11 LC8-peptide crystal structures that were used to generate the sequence logo in Figure 1b.
- https://doi.org/10.7554/eLife.36258.004
Figure 2 with 1 supplement
Domain structures of dASCIZ.
(a) Domain structure of dASCIZ, showing the dZnF domain (red) and 7 LC8 binding motifs in its C-terminal domain (blue). Dark blue bars indicate predicted TQT motifs and gray bars indicate the TMT …
-
Figure 2—source data 1
Tables containing the NMR data that were used to generate the graphs in Figure 2e–g.
- https://doi.org/10.7554/eLife.36258.007
Figure 2—figure supplement 1
dLBD C-terminus is transiently compact.
(a) Far UV CD spectrum of dLBD constructs collected at 10°C: QT1-3 (red), QT2-4 (orange), QT4-6 (green), and QT4-7 (magenta). (b) Size exclusion chromatography of each construct from (a) depicts …
Figure 3
LC8-dLBD interactions monitored by ITC.
(a) Construct schematics of the dLBD, QT1-3, QT2-4, QT4-6, and QT4-7 are shown, along with the locations of each TQT motif. A representative isothermal titration calorimetry thermogram of LC8 with …
Figure 4 with 1 supplement
ASCIZ and LC8 form a dynamic complex with low occupancy intermediates.
(a) Representative c(S) distributions obtained by sedimentation velocity are shown for the dLBD, LC8, and increasing molar ratios of the dLBD: LC8; 1:1, 1:3, 1:6, and 1:10. The standardized …
Figure 4—figure supplement 1
SAXS data of dLBD:LC8 complex.
(a) A Guinier plot of the experimental scattering data is shown. The solid line represents the Guinier fit, where the linear fit was extended to q < 1.3/Rg (Kikhney and Svergun, 2015). (b) The …
Figure 5 with 1 supplement
dLBD:LC8 and hLBD:LC8 complexes visualized by negative stain electron microscopy.
(a) Representative micrograph of negatively stained hLBD:LC8 complexes. Identified oligomeric complexes are boxed. Non-oligomeric LC8 dimers are indicated by arrowheads. Scale bar = 100 nm. dLBD:LC8 …
Figure 5—figure supplement 1
Single particle images of dLBD:LC8 and hLBD:LC8 complexes.
(a) Representative images of negatively stained (a) dLBD:LC8 complexes and (b) hLBD:LC8 complexes extracted from raw micrographs. Each particle is annotated to indicate the assigned occupancy of LC8 …
Figure 6 with 1 supplement
NMR titration of the dLBD with LC8.
Relative intensities of non-proline NH peaks in 15N-13C-HNCO spectra are shown for (a) dLBD, (b) QT2-4, and (c) QT4-6 titrated with LC8 at molar ratios of 1:0.25, 1:1, 1:2, 1:5, and 1:8. Peak …
-
Figure 6—source data 1
A table of the average I/I0 values for each 10-amino acid motif in the dLBD construct, QT2-4 construct, and QT4-6 construct.
These values were used to create the graphs in Figure 6e–g.
- https://doi.org/10.7554/eLife.36258.017
Figure 6—figure supplement 1
Representative HNCO slices of dLBD titration with LC8.
Shown are representative peaks from HNCO spectra depicting a loss of intensity with an increasing LC8 concentration. Colors are the same as Figure 6a: free dSQTQ (black), 1 LBD: 0.25 LC8 (red), 1:1 …
Figure 7 with 1 supplement
The number of LC8 recognition motifs tunes ASCIZ transcriptional activity.
(a) Domain structure of human ASCIZ, showing 11 LC8 binding motifs as blue bars. Additional non-TQT motifs are shown as gray bars. (b) Shown are representative isothermal titration plots of LC8 with …
-
Figure 7—source data 1
The raw data from the firefly luciferase reporter assays, shown for each ASCIZ construct.
- https://doi.org/10.7554/eLife.36258.020
Figure 7—figure supplement 1
Western blot of ASCIZ constructs
Western blot analysis of human U2OS cells transiently transfected with ASCIZ constructs from (Figure 7c), probed with ASCIZ antibody.
Figure 8
Model of ASCIZ regulation of LC8 transcription.
A proposed model of LC8 transcriptional regulation is shown for dASCIZ, which also applies to the human protein. Free LC8 dimers (dark blue) bind to ASCIZ and modulate transcriptional activity. …
Tables
Table 1
Thermodynamic parameters of dASCIZ-LC8 interactions.
https://doi.org/10.7554/eLife.36258.009| Construct | N | Overall Kd (μM) | Overall ΔH (kcal/mol) | Overall TΔS (kcal/mol) | Overall ΔG (kcal/mol) |
|---|---|---|---|---|---|
| dLBD | 7.3 | 1.4 ± 0.1 | −5.3 ± 0.2 | 2.7 ± 0.4 | −8.0 ± 0.4 |
| QT1-3 (241–324) | 3.2 | 2.4 ± 0.1 | −8.1 ± 0.4 | −0.4 ± 0.6 | −7.7 ± 0.4 |
| QT2-4 (271–341) | 2.7 | 4.1 ± 0.2 | −7.9 ± 0.4 | −1.6 ± 0.5 | −6.3 ± 0.3 |
| QT4-6 (321–376) | 3.0 | 1.0 ± 0.1 | −10.0 ± 0.5 | −1.8 ± 0.6 | −8.2 ± 0.4 |
| QT4-7 (321–388) | 4.0 | 1.6 ± 0.4 | −10.0 ± 0.5 | −2.1 ± 0.6 | −7.9 ± 0.4 |
Table 2
Thermodynamic parameters of peptide-LC8 interactions.
https://doi.org/10.7554/eLife.36258.010| Peptide | Peptide Sequence*,† | N | Kd (μM) | ΔH (kcal/mol) | TΔS (kcal/mol) | ΔG(kcal/mol) |
|---|---|---|---|---|---|---|
| QT1p | ymssQKLDMETQTEe | 1.1 | 14 ± 3.5 | −5.9 ± 0.3 | 0.7 ± 0.4 | −6.6 ± 0.3 |
| QT2p | ylapLLRDIETQTPd | 1.0 | 7 ± 0.4 | −9.2 ± 0.5 | −2.2 ± 0.6 | −7.0 ± 0.4 |
| QT3p | ytpdTRGDIGTMTDd | --- | weak | ----- | ----- | ----- |
| QT4p | dlqTSAHMYTQTCd | 1.1 | 15 ± 0.8 | −8.7 ± 0.4 | −2.1 ± 0.5 | −6.6 ± 0.3 |
| QT5p | eelGLSHIQTQTHw | 0.9 | 11 ± 0.6 | −8.8 ± 0.4 | −2.0 ± 0.5 | −6.8 ± 0.3 |
| QT6p | wpdgLYNTQHTQTCd | 1.1 | 20 ± 1.0 | −8.6 ± 0.4 | −2.2 ± 0.5 | −6.4 ± 0.3 |
| QT7p | epdNFQSTCTQTRw | 1.1 | 10 ± 0.5 | −7.8 ± 0.4 | −0.9 ± 0.5 | −6.9 ± 0.3 |
-
*the 10-amino acid LC8 binding motif is capitalized
†non-native residues added to the N-terminus of each peptide to increase solubility or improve concentration determination are underlined
Table 3
Thermodynamic parameters of ASCIZ-LC8 interactions.
https://doi.org/10.7554/eLife.36258.021| Construct | N | Overall Kd (μM) | Overall ΔH (kcal/mol) | Overall TΔS (kcal/mol) | Overall ΔG (kcal/mol) |
|---|---|---|---|---|---|
| hLBD | 11.2 | 0.9 ± 0.1 | −10.6 ± 0.5 | −2.4 ± 0.6 | −8.2 ± 0.4 |
| AAA8-11 | 6.7 | 0.7 ± 0.1 | −10.4 ± 0.5 | −2.0 ± 0.6 | −8.4 ± 0.4 |
| AAA1-4 | 6.6 | 2.7 ± 0.1 | −12.6 ± 0.6 | −5.0 ± 0.7 | −7.6 ± 0.4 |
| AAA5-11 | 4.2 | 1.5 ± 0.1 | −9.2 ± 0.5 | −1.3 ± 0.6 | −7.9 ± 0.4 |
| AAA1-4, 8–11 | 2.5 | 4.4 ± 0.2 | −12.2 ± 0.6 | −4.9 ± 0.7 | −7.3 ± 0.4 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | ASCIZ | NA | Uniprot ID: O43313 | |
| Gene (Drosophila melanogaster) | dASCIZ | NA | Uniprot ID: Q9VZU1 | |
| Cell line (Mus musculus) | ASCIZ knockout mouse embryonic fibroblast | PMID: 22167198 | ||
| Antibody | ASCIZ (rabbit monoclonal) | PMID: 15933716 | WB: 100 ng/mL | |
| Commercial assay or kit | Dual-luciferase reporter assay kit | Promega | Catalog number: E1910 | |
| Chemical compound, drug | D-glucose 13C6 | Sigma Aldrich | Catalog number: 389374 | |
| Chemical compound, drug | Ammonium-15N chloride | Sigma Aldrich | Catalog number: 299251 | |
| Software, algorithm | Origin 7.0 | OriginLab | ||
| Software, algorithm | SEDFIT | open-source | ||
| Software, algorithm | SEDNTERP | open-source | ||
| Software, algorithm | SEDPHAT | open-source | ||
| Software, algorithm | Curvefit | Palmer lab website | ||
| Software, algorithm | Topspin | Bruker Biospin Corporation | RRID:SCR_014227 | |
| Software, algorithm | Sparse Multidimentional Fourier Transform | Kozminski lab website | ||
| Software, algorithm | NMRFAM-Sparky | NMR facility at University of Wisconsin-Madison website | RRID:SCR_014228 | |
| Software, algorithm | ATSAS package | EMBL Hamburg BioSAXS website | RRID:SCR_015648 | |
| Software, algorithm | EMAN2 | National Center for Macromolecular Imaging | ||
| Software, algorithm | RELION 2.0 | open-source |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.36258.023
