Fig. 4: Analysis of active APC/C-dependent ubiquitination architecture reveals unexpected Ub binding modes.
a, Focused 3D classification on CDH1, UBE2C and APC2/APC11 using a mask to generate structures of the active APC/C. Discrete states were found showing a Ub interacting with the known APC11 RING exosite (left) and forming contacts with CDH1 (center left) and UBE2C (center right). A class of particles also contained a structure with two Ubs making simultaneous contact with the coactivator and UBE2C simultaneously (right). b, Phage display selected a UbV (UbVCDH1) that binds to APC/C coactivators. c, Sequence alignment of Ub and UbVCDH1 with differences highlighted. d, Coomassie-stained SDS–PAGE gel showing binding of GST–UbVCDH1 towards APC/C coactivators (CDH1 and CDC20), but not GST–Ub, after GST pulldown. n = 3 independent experiments. e, Degradation of APC/C substrates Cyclin A, Cyclin B, Geminin and Securin in mitotic HeLa cell extracts was inhibited by the addition of UbVCDH1, but not Ub, as monitored by immunoblotting. n = 3 independent experiments. f, Attachment of the Hsl1 D-box, but not the Hsl1 KEN-box, to UbVCDH1 potentiates its inhibition of APC/C–CDH1–UBE2C-mediated substrate ubiquitination, monitored in three independent experiments by fluorescent scanning of an SDS–PAGE gel. g, Left: the cryo-EM structure of APC/C-CDH1 bound to the UbVCDH1(orange)–Hsl1 D-box (red) shows the localization of UbVCDH1 near the KEN-box-binding site on the CDH1 β-propeller (purple). Right: previously published cryo-EM map of APC/C bound to CDH1 and Hsl1 (EMD-2651) for comparison28. Uncropped gels representative of n = 3 independent experiments for d–f available in source data.
