Oxidized and X-ray-reduced Apoptosis-Inducing Factor with Allosteric Fragment Binders Examined by Time-resolved HT-SAXS

This dataset applies time-resolved HT-SAXS to identify and triage allosteric fragment binders of oxidized and X-ray-reduced Apoptosis-Inducing Factor (AIF). Fragment hits of AIF were identified by high-throughput differential scanning fluorescence (HT-DSF), then validated by DSF and microscale thermophoresis (MST). SAXS data with 300-ms framing were collected on 39 AIF-fragment complexes and analyzed at 0.3-s and 2.1-s exposures to determine fragment impact on oxidized and X-ray-reduced AIF redox states, respectively. These datasets revealed fragments capable of stimulating dimerization of X-ray-reduced AIF and triaged fragment binders into conformational chemotypes. This dataset is described in detail in Chemical screening by time-resolved X-ray scattering to discover allosteric probes. Brosey CA, Link TM, Shen R, Moiani D, Burnett K, Hura GL, Jones DE, Tainer JA. Nature Chemical Biology DOI: 10.1038/s41589-024-01609-1.

N-terminally truncated AIF (78-613) was expressed in bacteria and purified as described in Brosey et al, Structure, 2016. Purified AIF was exchanged into SAXS buffer by size-exclusion chromatography (Superdex 200 10/300) to ensure monodisperse protein. SAXS samples were prepared at 4 mg/mL with direct addition of DMSO or library fragments (10 mg/mL) to protein and SEC-matched buffers (Brosey et al, Methods in Enzymology, 2023). HT-SAXS data were collected by mail-in service at ALS beamline 12.3.1 (SIBYLS) at Lawrence Berkeley National Laboratory. **For deposition, this dataset was reprocessed and rescaled from the original image files to ensure correct intensity errors.**

Two zip files containing 0.3 s (oxidized) and 2.1 s (X-ray-reduced) SAXS curves for AIF-fragment complexes. File names are descriptive of DMSO or fragment identifier.

2023-03-23

2024-05-30

HT-SAXS

Advanced Light Source

SIBYLS BL12.3.1

1.27 Å

2.0 m

Sample:

• Macromolecule 1: AIF
• Sample Full Name: Apoptosis-inducing Factor
• Sample Type: Protein
• Source Organism: Homo sapiens
• Source Organism NCBI Taxonomy ID: 9606
• Expression System: Escherichia coli
• Expression NCBI Taxonomy ID: 562
• Uniprot ID: O95831
• Sequence or Chemical Formula:
• MVGAGAYAYKTMKEDEKRYNERISGLGLTPEQKQKKAALSASEGEEVPQDKAPSHVPFLLIGGGTAAFAAARSIRARDPGARVLIVSEDPELPYMRPPLSKELWFSDDPNVTKTLRFKQWNGKERSIYFQPPSFYVSAQDLPHIENGGVAVLTGKKVVQLDVRDNMVKLNDGSQITYEKCLIATGGTPRSLSAIDRAGAEVKSRTTLFRKIGDFRSLEKISREVKSITIIGGGFLGSELACALGRKARALGTEVIQLFPEKGNMGKILPEYLSNWTMEKVRREGVKVMPNAIVQSVGVSSGKLLIKLKDGRKVETDHIVAAVGLEPNVELAKTGGLEIDSDFGGFRVNAELQARSNIWVAGDAACFYDIKLGRRRVEHHDHAVVSGRLAGENMTGAAKPYWHQSMFWSDLGPDVGYEAIGLVDSSLPTVGVFAKATAQDNPKSATEQSGTGIRSESETESEASEITIPPSTPAVPQAPVQGEDYGKGVIFYLRDKVVVGIVLWNIFNRMPIARKIIKDGEQHEDLNEVAKLFNIHEDLEVLFQ