Extended Data Fig. 5: E2 recruitment by CUL9-RBX1.
a, Isothermal titration calorimetry of CUL9ARIH-RBR-RBX1 with UBE2D2, UBE2D3 and UBE2L3. b, Comparison of proteins identified by mass spectrometry as interacting with TwinStrep-CUL9-RBX1 versus CUL9ΔRING1-RBX1 (deletion mutant of CUL9’s RING1 domain) expressed in HEK293S cells. Volcano plots of p-values (-log10) from two-tailed Student’s t tests versus protein abundance (log2) differences. The significance curve was calculated based on a false-discovery-rate-adjusted P = 0.01 and a minimal fold change S0 = 0.6. Proteins above the curve show significant differences between CUL9-RBX1 and CUL9ΔRING1-RBX1. The ubiquitin E2 enzymes identified are highlighted in blue and CUL9 and UBE2L3 highlighted and labeled in red. Data were obtained for each protein from three independent biological replicates. c, Overlay of E3 RING1 domain and E2 from ARIH1-UBE2L3 complex (PDB:7B5L), CUL9-RBX1 RING1 domain and the E2 from this study, and an AlphaFold2 model of the CUL9 RING1 domain aligned on the RING1 domains. d, Crosslinks between UBE2L3 and CUL9 ARM3 domain mapped onto the CUL9-RBX1 cullin dimer structure and a modeled neighboring protomer. e, Visualization of BS3 cross-linking mass spectrometry analysis of CUL9-RBX1 sample mixed with UBE2L3~ubiquitin. 2D Plots were visualized with XiNET (www.crosslinkviewer.org). Table of crosslinks can be found in Supplementary Table 1.
