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Open Dataset XSUTRZQL


Apoptosis-inducing factor (AIF) allostery mutants and their impact on NADH-driven dimer formation

Abstract

Oxidoreductase Apoptosis-inducing factor (AIF) supports critical mitochondrial activities, including OXPHOS biogenesis and cristae remodeling, and participates in the PARP-1-dependent cell death pathway parthanatos. These functions are seemingly regulated by allosteric monomer-dimer switching driven by binding and reduction by NADH. Using small-angle X-ray scattering (SAXS), we characterized a panel of monomer and dimer-permissive mutants to define molecular pathways linking AIF’s NADH active site to allosteric switching regions. These results were combined with X-ray crystallography and molecular dynamics and reported in “Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor". Brosey CA, Ho C, Long WZ, Singh S, Burnett K, Hura GL, Nix JC, Bowman GR, Ellenberger T, Tainer JA. Structure, 6:2067-2079 (2016). DOI: 10.1016/j.str.2016.09.012.

Experimental description

N-terminally truncated AIF (78-613) and point mutants were expressed in bacteria and purified by nickel affinity and gel filtration chromatography (expression tags were removed post-nickel purification). Purified proteins were concentrated and exchanged into SAXS buffer by size-exclusion chromatography (Superdex 200 10/300) to ensure monodisperse protein. SAXS samples were prepared at 3-4 mg/mL with direct addition of NADH ligand to protein and SEC-matched buffers. HT-SAXS data were collected by mail-in service at ALS beamline 12.3.1 (SIBYLS) at Lawrence Berkeley National Laboratory. The dataset available here contains wild-type AIF (78-613) and point mutants collected at 3-4 mg/mL with and without 10-fold molar excess NADH. Additional I(q) curves include the AIF-ΔC-loop mutant (C-loop dataset), which replaces AIF’s allosteric C-terminal loop (residues 511-558) with a GGGSSS insert, and a second truncation mutant (AIF121 datasets, residues 121-613) that completely removes AIF’s flexible N-terminus.

File description

File names are descriptive. Wild-type AIF is designated as WT. N-terminal truncations are denoted as AIF78 (residues 78-613) or AIF121 (residues 121-613). Point mutations or the C-loop deletion are as indicated in the AIF78 background. The presence of NADH is also noted.

Created

2022-07-14

Published

2024-03-30

Data collection technique

HT-SAXS

Journal DOI

https://doi.org/10.1016/bs.mie.2022.09.024

Source

Advanced Light Source

Beamline

SIBYLS BL12.3.1

Wavelength

1.03 Å

Sample to Detector Distance

1.5 m


Submitting Author

Chris Brosey

University of Texas MD Anderson Cancer Center

United States of America

[email protected]

Collaborators

  • Greg Hura, Lawrence Berkeley National Laboratory
  • Kathryn Burnett, Lawrence Berkeley National Laboratory

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.

  • AIFmutants-WithORWithout_NADH.zip Download


Individual SAS Data Files (total 1)

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Supplemental Data and Supporting Materials (total 2)

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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Sample:

  • Macromolecule 1: AIF
    • Sample Full Name: Apoptosis-Inducing Factor
    • Sample Type: Protein
    • Source Organism:
    • Source Organism NCBI Taxonomy ID:
    • Expression System:
    • Expression NCBI Taxonomy ID:
    • Uniprot ID's:
    • Sequence or Chemical Formula: