Fig. 2: Structural arrangements of SIFI modules. | Nature

Fig. 2: Structural arrangements of SIFI modules.

From: Molecular basis of SIFI activity in the integrated stress response

Fig. 2

a, Calmodulin (green; CaM) binds to the outer rim of the SIFI scaffold above the C-terminal UBR4 dimerization interface. b, The N-terminal lobe of calmodulin (green) forms a helix bundle with UBR4 (light grey), while the Ca2+-bound C-terminal lobe of calmodulin engages a calmodulin-binding motif of UBR4 (gold), situated atop the UBR4 lid (blue grey) that encircles the C-terminal α-helix of KCMF1 (plum). Conserved residues involved in the interaction are highlighted within dashed boxes. c, Calmodulin stabilizes the base of the UBR4 C-terminal catalytic arm, including hemi-RING, UBE2A (coral) and UBL (blue), allowing flexible movement of the arm around residue Gly4301 (red dot) of UBR4. d, Two copies each of SIFI’s protein interaction modules (coloured) are located at the centre of the scaffold (left). Right, magnified view of the DOC2 (brown) and WD40 (teal) domains, and the KCMF1N138–DOC1–UBR subcomplex (plum, blue and yellow). e, The N-terminal ZZ domain of KCMF1 is required for ubiquitylation activity. WT or ΔZZ KCMF1 were purified from ΔUBR4 cells to prevent UBR4-dependent ubiquitylation and incubated with E1, E2 (UBE2D3, UBE2A) and ubiquitin. Activity was detected by formation of high-molecular-mass conjugates using anti-ubiquitin western blotting. Ubiquitylated species (UBI). Similar results were observed in n = 2 experiments. f, C-terminal helix of KCMF1 (plum) is anchored within α-helical bundles of UBR4’s armadillo repeats (light grey) through the UBR4 lid (blue grey). A conserved Phe319 of KCMF1 (plum), surrounded by three Trp residues of UBR4, is highlighted. g, Deletion or mutation (R316E/F319E/L323E/L325E) of the C-terminal helix (CTH mutant) impedes KCMF1 integration into SIFI, as shown by KCMF1–Flag affinity purification and detection of UBR4. A Q226/228/229/231/233E mutation in an unresolved linker of KCMF1 (mutLINK) did not affect integration into SIFI. ABHD10 binds to the ZZ domain of KCMF1 and was used as a positive control. Similar results were observed in n = 2 experiments. Gel source data are provided in Supplementary Fig. 1.

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