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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.

Data collection technique:

HT-SAXS

Wavelength

Sample to Detector Distance


Created: 2022-07-14

Updated: 2022-07-15

Submitting Author:

Chris Brosey

University of Texas MD Anderson Cancer Center

United States of America

[email protected]

Collaborators:

Project Leader:

John Tainer

[email protected]


Data Download Terms:

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Complete Set of SAXS Data Files:

The complete SAXS dataset is downloadable as a zip file.

  • AIFmutants-WithORWithout_NADH.zip Download


Sample SAXS Data Files (total 1):

These are individual SAXS data files that are included in the complete dataset, but are available separately for quick download as examples of what is contained in the zip file.


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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SAXS Similarity SAXS FrameSlice


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:
    • Sequence or Chemical Formula: