Extended Data Fig. 4: Related to Fig. 4.
From: PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase
a, Model of the AURORA-A (blue) / CEREBLON (purple) complex. Structures of AURORA-A with alisertib and of CEREBLON with lenalidomide39 were used for protein-protein docking. The top-ranking solution is displayed (ACc1: AURORA-A-CEREBLON complex 1). Alisertib and lenalidomide are shown in green and aqua, respectively. b, Model of the second-best AURORA-A (blue)-CEREBLON (purple) complex compatible with JB170 binding (ACc2). Structures of AURORA-A with alisertib and of CEREBLON with lenalidomide were used for protein-protein docking. Alisertib and lenalidomide are shown in green and aqua, respectively. c, Modeled ternary complex structure of JB170 (orange) bound to the ACc1 shown in Extended Data Fig. 4a. Alisertib (green) and lenalidomide (aqua) were modified, connected and minimized to give JB170. d, Modeled ternary complex of JB170 (orange) bound to the ACc2 shown in Extended Data Fig. 4b. Alisertib (green) and lenalidomide (aqua) were modified, connected and minimized to give JB170. e, Docking solutions of the active degraders JB170 and JB158 as well as the less functional degraders JB159, JB169, JB171 and negative control JB211 in the AURORA-A (blue)-CEREBLON (purple) complexes ACc1 and ACc2. Scaffolds of thalidomide and alisertib used as light constraints during docking and as reference structures for the substructure root-mean-square deviation of atomic positions (RMSD) measurements are shown. Active degraders (labeled in green) achieve RMSD values below 1 Å with respect to both alisertib and the thalidomide scaffold. f, Immunoblots of immunoprecipitation experiments. Cells expressing HA-tagged wild-type AURORA-A (WT), a version of AURORA-A (AURORA-AImut) with 12 amino acid substitutions (R137E, K153E, K156E, F157E, I158E, R189E, P191W, K224E, E239R, S266W, A267W and R375E) or an empty vector (Ctr) were treated with 0.5 µM JB170 for 6 h. AURORA-A was precipitated with an anti-HA tag antibody, and the amount of co-precipitated CEREBLON was tested by immunoblotting. g, AURORA-A levels based on luciferase measurements. Wild-type (WT) or AURORA-A with one amino acid substitution (AURORA-AP191W) were fused to luciferase fragment (HiBiT) and expressed in MV4–11 cells. Cells were treated with 1 µM alisertib for 6 h, lysed, complemented with the second luciferase fragment (largeBiT), and measured for luciferase activity. Bars represent mean ± s.d. of n = 3 replicates.
