Journal: iScience
Article Title: Structural basis of nucleosome deubiquitination by the bidentate Calypso/Asx complex
doi: 10.1016/j.isci.2026.114958
Figure Lengend Snippet: The second Calypso/Asx heterodimer facilitates efficient nucleosome processing (A) Comparison of the electron density maps of Calypso/Asx/NCP-ub and BAP1/ASXL1/NCP-ub, with the nucleosome positioned at the same angle. The resolution of BAP1/ASXL1/NCP-ub has been low-pass filtered. (B) Previously proposed ubiquitin-binding site and key residues are shown (doi, https://doi.org/10.1038/s41467-018-06186-1 ). (C) A cartoon representation illustrates that, building upon the established binding mode of Calypso-1/Asx-1 with nucleosome-ub-1, a second nucleosome-ub-2 was modeled onto the catalytic center of Calypso-2/Asx-2 using the binding configuration observed for Calypso-1/Asx-1 with nucleosome-ub-1. This modeling results in pronounced steric clashes in the nucleosomal region (indicated by red dashed lines). Subunit coloring is maintained consistently with the previous figures. (D) A close-up view showing the key amino acids of the two Calypso coiled-coil hairpins, with the model referenced from PDB: 6CGA . (E) SEC-MALS analysis of wild-type Calypso/Asx (1–340) (dimer) and mutant Calypso/Asx (1–340) (monomer), with the following mutations in Calypso: M288R, N292R, L340R. The gel filtration column is Superdex 200 5/150. (F) Michaelis-Menten analysis of wild-type and mutant Calypso/Asx proteins cleavage of ubiquitin-AMC. Error bars indicate ±SEM ( n = 3 independent experiments). (G) Activity assays comparing the ability of wild-type (dimer), mutant (monomer) Calypso/Asx complexes to remove ubiquitin from nucleosomes mono-ubiquitinated at H2AK119. The H3 band was run on the same gel as a control.
Article Snippet: Superdex 200 Increase 10/300 GL , Cytiva , Cat#28990944.
Techniques: Comparison, Ubiquitin Proteomics, Binding Assay, Mutagenesis, Filtration, Activity Assay, Control
