Extended Data Fig. 2: Structural domains of SIFI.
From: Molecular basis of SIFI activity in the integrated stress response
a. Structural insights into the N-terminal dimerization region of SIFI. Left: two Lys residues at the tips of interlocking loops from each protomer engage in polar interactions with their paired counterpart. Right: α7 helix of UBR4 undergoes a helix swap with the paired protomer to form a helical bundle. b. C-terminal dimerization region. 180° rotation reveals the stabilizing helical pair at the inside of the SIFI scaffold. c. The SIFI scaffold shows flexibility around a central hinge region, leading to ~10° rotations of the N- and C-terminal dimerization domains relative to each other. Two disease mutations in UBR4, A2581 (ataxia) and R2584 (cancer) are located close to the hinge region. d. Calcium-binding to the C-lobe, but not the N-lobe of calmodulin is required for binding of UBR4, as seen by immunoprecipitation of calmodulin mutants that disrupt calcium-binding in the N- or C-lobe, respectively (N-lobe mutant: D21A, D23A, D57A, D59A; C-lobe mutant: D94A, D96A, D130A, D132A)28. Similar results in n = 2 independent experiments. e. Cryo-EM map of central SIFI showing docked WD40, DOC and UBR domains. These internal protein interaction modules are flexible and hence show lower overall resolution. The central density (ABHD10) is described below. f. The WD40 domain of UBR4 features a central plug helix at a surface commonly involved in protein interactions characteristic of WD40 repeats. g. The DOC2 domain of UBR4 contains two Zinc-binding loops that collectively form a potential protein interaction interface. h. Diagram of cross-linking mass spectrometry validates domain arrangement at the centre of SIFI. For gel source data, see Supplementary Fig. 1.
