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Open Dataset XS4FJZBK

CHCHD4 Chaperone and AIF-interaction Mutants

Abstract

This dataset reports X-ray scattering profiles for a panel of CHCHD4 mutants targeting the N-terminal AIF-interaction motif. SAXS analysis indicates that CHCHD4 mutants most defective for AIF interaction exhibit a more extended architecture relative to the wild-type protein. Complementary NMR studies ultimately revealed that the N-terminal interaction domain contacts and shields CHCHD4’s central catalytic domain; thus mutations to the N-terminus interfere with this intramolecular regulatory interaction. Additional analyses for these SAXS data may be found at the SASBDB with entries SASDVY6 (CHCHD4-WT), SASDVY6 (I12A/F14A/H20A), SASDV27 (H20D), SASDV37 (L28D). This dataset is described in further detail in Brosey CA, Shen R, and Tainer JA, The EMBO Journal, 2025.

Experimental description

Wild-type and mutant CHCHD4 constructs were expressed and purified as described in Brosey et al, Nature Chemical Biology, 2024. Purified wild-type and mutant CHCHD4 were exchanged into SAXS buffer (25 mM HEPES, 150 mM NaCl, 2 mM TCEP, pH 7.5) by size-exclusion chromatography (Superdex 75 10/300) to ensure monodisperse protein. Peak CHCHD4 fractions from Superdex 75 purifications were pooled (1.5-2.0 mL), concentrated to 3-7.3 mg/mL in 4-mL Amicon ultra centrifugal filters concentrators with 10-kDa molecular-weight cut-off limits (Millipore-Sigma), then diluted into concentration series of 0.6-7.3 mg/mL. Samples were transferred into 96-well PCR plates with matched buffers eluted from the protein-free void volume or collected from concentration flowthroughs. Plates were flash frozen in liquid nitrogen and shipped on dry ice for mail-in HT-SAXS data collection at ALS beamline 12.3.1 (SIBYLS) at Lawrence Berkeley National Laboratory.

File description

The zip file contains scattering curves (averaged across 0.3 s frames by SAXS FrameSlice) for wild-type CHCHD4 (CHCHD4-WT.dat) and mutants I12A/F14A/H20A (CHCHD4-AIA-A.dat), H20D (CHCHD4-H20D.dat), and L28D (CHCHD4-L28D.dat).

Created

2024-03-30

Published

2025-03-04

Data collection technique

HT-SAXS

Journal DOI

https://doi.org/10.1038/s44318-024-00360-6

Source

Advanced Light Source

Beamline

SIBYLS BL12.3.1

Wavelength

1.23 Å

Sample to Detector Distance

2.0 m

Submitting Author

Chris Brosey

University of Texas MD Anderson Cancer Center

United States of America

[email protected]

Collaborators

Project Leader

John Tainer

[email protected]


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Complete Set of SAS Data Files

The complete SAS dataset is downloadable as a zip file.

Individual SAS Data Files (Total: 0)

These are individual SAS data files that may be raw or processed (merged, etc.). These may or not be included in a zip file containing a larger dataset.

Supplemental Data and Supporting Materials (Total: 0)

These data and materials may include X-ray crystal structure coordinates, multi-angle light scattering data, .etc. It may also include additional details or methods pertaining to the SAXS experiments, or the researcher's interpretations of the results.

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SAXS Similarity SAXS FrameSlice

Samples:

  • Macromolecule 1: CHCHD4
    • Sample Full Name: Coiled-coil-helix-coiled-coil-helix domain
    • Sample Type: Protein
    • Source Organism: Homo sapiens
    • Source Organism NCBI Taxonomy ID: 1758
    • Expression System: Escherichia coli
    • Expression NCBI Taxonomy ID: 562
    • Uniprot ID: Q8N4Q1
    • Sequence or Chemical Formula:
    • GPMSYCRQEGKDRIIFVTKEDHETPSSAELVADDPNDPYEEHGLILPNGNINWNCPCLGGMASGPCGEQFKSAFSCFHYSTEEIKGSDCVDQFRAMQECMQKYPDLYPQEDEDEEEEREKKPAEQAEETAPIEATATKEEEGSS
  • Macromolecule 2: CHCHD4-I12A-F14A-H20A
    • Sample Full Name: Coiled-coil-helix-coiled-coil-helix domain I12A/F14A/H20A
    • Sample Type: Protein
    • Source Organism: Homo sapiens
    • Source Organism NCBI Taxonomy ID: 1758
    • Expression System: Escherichia coli
    • Expression NCBI Taxonomy ID: 562
    • Uniprot ID: Q8N4Q1
    • Sequence or Chemical Formula:
    • GPMSYCRQEGKDRAIAVTKEDAETPSSAELVADDPNDPYEEHGLILPNGNINWNCPCLGGMASGPCGEQFKSAFSCFHYSTEEIKGSDCVDQFRAMQECMQKYPDLYPQEDEDEEEEREKKPAEQAEETAPIEATATKEEEGSS
  • Macromolecule 3: CHCHD4-H20D
    • Sample Full Name: Coiled-coil-helix-coiled-coil-helix domain H20D
    • Sample Type: Protein
    • Source Organism: Homo sapiens
    • Source Organism NCBI Taxonomy ID: 1758
    • Expression System: Escherichia coli
    • Expression NCBI Taxonomy ID: 562
    • Uniprot ID: Q8N4Q1
    • Sequence or Chemical Formula:
    • GPMSYCRQEGKDRIIFVTKEDDETPSSAELVADDPNDPYEEHGLILPNGNINWNCPCLGGMASGPCGEQFKSAFSCFHYSTEEIKGSDCVDQFRAMQECMQKYPDLYPQEDEDEEEEREKKPAEQAEETAPIEATATKEEEGSS
  • Macromolecule 4: CHCHD4-L28D
    • Sample Full Name: Coiled-coil-helix-coiled-coil-helix domain L28D
    • Sample Type: Protein
    • Source Organism: Homo sapiens
    • Source Organism NCBI Taxonomy ID: 1758
    • Expression System: Escherichia coli
    • Expression NCBI Taxonomy ID: 562
    • Uniprot ID: Q8N4Q1
    • Sequence or Chemical Formula:
    • GPMSYCRQEGKDRIIFVTKEDHETPSSAEDVADDPNDPYEEHGLILPNGNINWNCPCLGGMASGPCGEQFKSAFSCFHYSTEEIKGSDCVDQFRAMQECMQKYPDLYPQEDEDEEEEREKKPAEQAEETAPIEATATKEEEGSS