Multivalency, autoinhibition, and protein disorder in the regulation of interactions of dynein intermediate chain with dynactin and the nuclear distribution protein

  1. Kayla A Jara
  2. Nikolaus M Loening
  3. Patrick N Reardon
  4. Zhen Yu
  5. Prajna Woonnimani
  6. Coban Brooks
  7. Cat H Vesely
  8. Elisar J Barbar  Is a corresponding author
  1. Department of Biochemistry and Biophysics, Oregon State University, United States
  2. Department of Chemistry, Lewis & Clark College, United States
  3. Oregon State University NMR Facility, United States
10 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Domain architecture for dynein intermediate chain (IC), dynactin p150Glued, and nuclear distribution protein (NudE).

(A) Domain architecture diagrams for IC from Drosophila melanogaster (Dros. IC) and Rattus norvegicus (Rat IC2C) and constructs used in earlier work are provided for comparison. Proteins and …

Figure 1—figure supplement 1
Intermediate chain (IC) sequence alignment.

Alignment of the first 260 residues of IC from Drosophila, human, and Chaetomium thermophilum using the Multiple Alignment using Fast Fourier Transform (MAFFT) alignment program (Katoh and Standley, …

Chaetomium thermophilum (Ct) p150CC1B, NudECC, and dynein light chains are dimeric, whereas Ct IC1-260 is monomeric.

(A) Sedimentation velocity analytical ultracentrifugation profiles for LC8 (red), LC7 (yellow), and Tctex (orange) (top), and, IC1-260 (gray), p150CC1B (blue), and NudECC (green) (bottom). All …

Figure 2—source data 1

Source files for sedimentation velocity analytical ultracentrifugation, sedimentation equilibrium analytical ultracentrifugation, circular dichroism, and size exclusion chromatography with multi-angle light scattering data.

This Excel workbook contains all the data plotted in Figure 2. The different sheets correspond to different panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig2-data1-v2.zip
Figure 3 with 2 supplements
Secondary structure and thermal stability of Chaetomium thermophilum (Ct) intermediate chain (IC).

(A) Agadir prediction for IC1-260 showing the percent helicity by residue (purple). Shown above the plot is a schematic structure for IC1-260 with labels for single α-helix (SAH), H2, and H3 above …

Figure 3—source data 1

Source files for IC1-260 helicity prediction and CD data.

This Excel workbook contains all the data plotted in Figure 3. The different sheets correspond to different panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig3-data1-v2.zip
Figure 3—figure supplement 1
Predicted percent helicity in intermediate chain (IC) across species.

Residue-level percent helicity predictions generated using the Agadir algorithm for the first 260 amino acids of IC from Homo sapiens (human), Rattus norvegicus (rat), Danio rerio (zebrafish), Callor…

Figure 3—figure supplement 1—source data 1

Source file for helix prediction of intermediate chain across species.

This Excel workbook contains the data plotted for the helix predictions shown in Figure 3—figure supplement 1. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
Chaetomium thermophilum (Ct) IC1-88 and Ct IC100-260 binding by sedimentation velocity analytical ultracentrifugation (SV-AUC).

(A) Domain architecture diagrams of IC1-260, IC1-88, and IC100-260. (B) SV-AUC experiments of IC100-260 (gray), IC100-260 mixed with IC1-88 at a 1:2 molar ratio (black), and IC1-260 (gray dashes). (C

Figure 3—figure supplement 2—source data 1

Source file for sedimentation velocity analytical ultracentrifugation (SV-AUC) and size exclusion chromatography with multi-angle light scattering (SEC-MALS) of binding interactions between IC1-88 and IC100-260.

This Excel workbook contains the data plotted for the SV-AUC and SEC-MALS data shown in Figure 3—figure supplement 2 with different sheets corresponding to different panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig3-figsupp2-data1-v2.zip
Figure 4 with 1 supplement
Identification of disordered linkers of Chaetomium thermophilum IC1-260 using nuclear magnetic resonance spectroscopy.

(A) Plot showing the normalized peak volumes at 10°C in the 1H-15N TROSY spectrum (gray) and in the CLEANEX spectrum (orange) of the amides that could be assigned. Assigned residues are in black in …

Figure 4—source data 1

Source files for IC1-260 nuclear magnetic resonance (NMR) TROSY and CLEANEX data.

This Excel workbook contains the peak heights and volumes for residues within IC1-260 in the TROSY and CLEANEX NMR experiments collected at 10°C. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig4-data1-v2.zip
Figure 4—figure supplement 1
Nuclear magnetic resonance spectra of intermediate chain (IC) are unaffected by salt concentration.

1H-15N TROSY spectra of IC1-260 in 20 mM NaCl (black) overlaid with IC1-260 in 250 mM NaCl (pink) at both 10°C (left) and at 40°C (right).

Evidence of tertiary contacts between the N and C-termini within Chaetomium thermophilum IC1-260.

(A) Domain architecture diagram for IC1-260 with bars shown below corresponding to the IC100-260, IC1-88, and IC160-240 constructs. (B) 1H-15N TROSY overlays of free 15N-labeled IC1-88 (black) and 15

Figure 5—source data 1

Source files for nuclear magnetic resonance binding studies of intermediate chain (IC) fragments.

This Excel workbook contains the peak heights and volumes for residues within the 15N-labeled IC1-88 and 15N-labeled IC216-260 constructs upon binding of IC100-260, IC160-240, and 15N-labeled IC216-260 or 15N-labeled IC1-88. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig5-data1-v2.zip
Figure 6 with 2 supplements
Binding interactions of Chaetomium thermophilum intermediate chain (IC) to p150CC1B and NudECC.

(A) Sedimentation velocity analytical ultracentrifugation profiles for samples containing p150CC1B (blue dashed line), NudECC (green dashed line), IC1-88/p150CC1B complex (blue solid line), and IC1-8…

Figure 6—source data 1

Source files for sedimentation velocity analytical ultracentrifugation (SV-AUC) and nuclear magnetic resonance (NMR) binding studies of intermediate chain (IC) fragments with p150CC1B and NudECC.

This Excel workbook contains the data plotted for the SV-AUC experiments as well as the NMR peak volume ratios for binding studies between 15N-labeled IC1-88 and 15N-labeled IC37-88 with p150CC1B and NudECC. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig6-data1-v2.zip
Figure 6—figure supplement 1
Size exclusion chromatography with multi-angle light scattering (SEC-MALS) of IC1-88 and p150CC1B.

The estimated mass of p150CC1B from MALS is 49 kDa (light blue) while the addition of IC1-88 increases the mass to 66 kDa (dark blue) indicating a complex containing a p150CC1B dimer and two IC1-88

Figure 6—figure supplement 1—source data 1

This Excel workbook contains the data plotted for the size exclusion chromatography with multi-angle light scattering data shown in Figure 6—figure supplement 1.

Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig6-figsupp1-data1-v2.zip
Figure 6—figure supplement 2
Binding interactions of Chaetomium thermophilum IC37-88 to p150CC1B and NudECC.

ITC thermograms for p150CC1B titrated with IC37-88 (left) and NudECC titrated with IC37-88 (right), collected at 25°C (pH 7.5). IC37-88 contains the H2 region of intermediate chain (IC) and binds …

Binding characterization of binary complexes of IC1-260.

(A) Sedimentation velocity analytical ultracentrifugation of IC1-260/LC8, IC1-260/Tctex, IC1-260/LC7, IC1-260/p150CC1B, and IC1-260/NudECC. Data for the binary complexes is overlayed with data for …

Figure 7—source data 1

Source files for sedimentation velocity analytical ultracentrifugation (SV-AUC) binding studies of IC1-260.

This Excel workbook contains the data plotted for the SV-AUC experiments shown in Figure 7. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig7-data1-v2.zip
Reconstitution and characterization of dynein subcomplexes.

(A) Size exclusion chromatography (SEC) traces of the dynein subcomplex (IC/light chains) (purple) and the dynein subcomplex with the addition of either p150CC1B (blue) or NudECC (green). (B) Sodium …

Figure 8—source data 1

Source files for size exclusion chromatography (SEC) and sedimentation velocity analytical ultracentrifugation (SV-AUC) of reconstituted IC1-260 subcomplexes.

This Excel workbook contains the data plotted for the SEC and SV-AUC experiments shown in Figure 8 with individual sheets corresponding to panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig8-data1-v2.zip
Figure 8—source data 2

Original sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel images.

This zipped folder contains the original files of the full raw unedited gel images for the size exclusion chromatography purification of each IC1-260 subcomplex. There is also a combined image with the uncropped gels with the relevant bands clearly labeled.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig8-data2-v2.zip
Binding characterization of Chaetomium thermophilum ICFL subcomplexes.

(A) The estimated mass of ICFL from multi-angle light scattering is 75.4 kDa, which indicates that ICFL is a monomer in the absence of binding partners. (B) Sedimentation velocity analytical …

Figure 9—source data 1

Source files for size exclusion chromatography with multi-angle light scattering (SEC-MALS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) of ICFL.

This Excel workbook contains the data plotted for the SEC-MALS experiment of ICFL and the SV-AUC binding experiments shown in Figure 9 with individual sheets corresponding to panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig9-data1-v2.zip
Figure 9—source data 2

Original sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel image.

This zipped folder contains the original file of the full raw unedited gel image for the IMAC fractions of ICFL. There is also an image with the uncropped gel with the relevant bands clearly labeled. Notice, two batches of ICFL were purified in tandem.

https://cdn.elifesciences.org/articles/80217/elife-80217-fig9-data2-v2.zip
A model of Chaetomium thermophilum IC1-260 binding interactions and subcomplex assemblies.

Free IC1-260 is compact and in autoinhibited state (Boxed in black), with the single α-helix (SAH), H2, and H3 regions depicted as helices and with colors indicating the LC8 (red), Tctex (orange), …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Chaetomium thermophilum)Ct ICNAUniProt: G0SCF1-1
Strain, strain background (Escherichia coli)Rosetta (DE3)Sigma-AldrichCatalog number: 70954
Cell line (Insect)Sf9ThermoFisherCatalog number:
11496015
Used within 1 yr of purchase
Strain, strain background (E. coli)DH10EMBacYGeneva BiotechDH10EMBacYUsed within 1 yr of purchase
Peptide, recombinant proteinCt IC full length proteinGenScriptpFastbac1 vector
Peptide, recombinant proteinIC1-88Ref. 32
Peptide, recombinant proteinIC37-88GenScript
Peptide, recombinant proteinIC160-240GenScript
Peptide, recombinant proteinIC216-260Azenta Life Sciences
Chemical compound, drugAmmonium- 15N chlorideSigma AldrichCatalog number: 299251
Chemical compound, drugD-glucose 13C6Sigma AldrichCatalog number: 389374
Software, algorithmOrigin 7.0OriginLab
Software, algorithmSEDFITOpen-source
Software, algorithmTopSpin 3.6Bruker Biospin CorporationRRID:SCR_014227
Software, algorithmCcpNmr analysis (CCPN)Ref. 80RRID:SCR_016984
Software, algorithmNMRPipeRef. 78

Additional files

Supplementary file 1

ITC results for intermediate chain interactions with p150CC1B and NudECC at 25°C.

aFor the p150CC1B experiments, a two-site binding model was used as it provided much better fits when compared to a single site model. bFitting of this experiment was done with set N values due to the amount of extra noise in the data and thus the N values are reported without error. cThis experiment did not show evidence of binding and thus the data was not fit to a binding model.

https://cdn.elifesciences.org/articles/80217/elife-80217-supp1-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/80217/elife-80217-mdarchecklist1-v2.docx

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