The carboxyl-terminal sequence of Bim enables Bax activation and killing of unprimed cells

Abstract

31The Bcl-2 family BH3 protein Bim promotes apoptosis at mitochondria by 32 activating the pore forming proteins Bax and Bak and by inhibiting the anti-apoptotic 33proteins Bcl-XL, Bcl-2 and Mcl-1. Bim binds to these proteins via its BH3 domain and to 34 the mitochondrial membrane by a carboxyl-terminal sequence (CTS). In cells killed by 35Bim, the expression of a Bim mutant in which the CTS was deleted (BimL-dCTS) 36triggered variable amounts of apoptosis that correlated with inhibition of anti-apoptotic 37proteins being sufficient to permeabilize mitochondria isolated from the same cells. 38Detailed analysis of the molecular mechanism demonstrated that BimL-dCTS inhibited 39Bcl-XL but did not activate Bax. Our examination of additional point mutants 40unexpectedly revealed that the CTS of Bim is required for physiological concentrations 41of Bim to activate Bax and that different residues in the CTS enable Bax activation and 42 binding to membranes. 43

Bim escapes displacement by BH3-mimetic anti-cancer drugs by double-bolt locking both Bcl-XL and Bcl-2

“…Moreover, the Bim CTS directly contributes to interactions with Bcl-XL (Figure 5 and Figure 7D–E) and much of this interaction is due to residue L185 (Figure 9). Consistent with this interpretation weak crosslinking was observed between this region of Bim and Bcl-XL (Chi et al, 2019). Thus unlike conventional lock and key models, the interaction of Bim with Bcl-XL and Bcl-2 is more reminiscent of a double-bolt lock with binding mediated by two distant interacting surfaces (separated by ~40 residues) working together.…”

“…Bim not only inhibits anti-apoptotic proteins it also activates the executioner proteins Bax and Bak, potentially confounding analysis. As this mechanism may be relevant in multiple cell lines it is examined in more detail elsewhere (Chi et al, 2019). Second, MCF-7 cells depend on MCL-1 for survival (Figure 3E).…”

“…Purified Bcl-XL and BimL protein binding in the presence of different concentrations of ABT-263 quantified in vitro using purified full-length proteins with ( D ) and without ( E ) liposomes. Control experiments demonstrating efficient binding of BimL to liposomes and Bcl-XL binding data for the Bim mutants are presented in the companion paper (Chi et al, 2019). Data are mean ±SD (n = 3 independent experiments).…”

“…Together the two anti-apoptotic protein binding sequences in Bim confer resistance of complexes to the dual Bcl-2, Bcl-XL inhibitor ABT-263 and to the new generation Bcl-XL inhibitors in clinical development. Because the CTS of Bim also functions to activate the pro-apoptotic proteins Bax and Bak, (Chi et al, 2019) our results suggest that BH3-mimetics may have unpredictable results in cells depending on the Bcl-2 family proteins expressed and that targeting both binding sites on Bcl-XL and/or Bcl-2 may be required to kill some cancer cells that have bypassed apoptosis by inhibiting Bim.…”

Allosteric Regulation of BH3 Proteins in Bcl-xL Complexes Enables Switch-like Activation of Bax

“…While we have established strong parallels between experiments using purified components and measurements of fluorescence protein fusions in live cells (Chi et al, 2019;Liu et al, 2019), limitations remain. The cell-based experiments required overexpression of the proteins.…”