Anti-apoptotic Bcl2 proteins, such as Bcl-XL and Bcl-2, modulate the intrinsic apoptosis pathway by sequestering pro-apoptotic Bcl2 proteins on the surface of mitochondria. Pro-apoptotic Bcl2 members, such as Bax and Bak, contain a semi-conserved, helical BH3 domain that binds to a shallow hydrophobic cleft on the surface of anti-apoptotic Bcl2 proteins. Unfortunately, non-specific interactions between pro- and anti-apoptotic Bcl2 members has complicated efforts to elucidate the mechanisms of Bcl2 signaling. Previous work by our lab has shown that the small protein scyllatoxin (ScTx) can be engineered to target anti-apoptotic Bcl2 proteins in vitro by grafting BH3 domain residues required for Bcl2 recognition to its α-helix. ScTx is a 31-amino acid protein that folds into an α/β structural motif stabilized by three disulfide linkages. Surprisingly, ScTx-BH3 domain mimetics designed to mimic the Bax BH3 domain (ScTx-Bax) containing three disulfides do not bind Bcl-2 in vitro, while fully-reduced ScTx-Bax variants bind Bcl-2 with sub-micromolar affinity. These results suggest that an induced-fit binding mechanism is required for favorable ScTx-Bax:Bcl-2 interactions. We have now expanded on these studies by designing a library of structural ScTx-Bax variants that vary in the number and position of disulfide linkages within their primary sequence. More specifically, we have developed a library of ScTx-Bax proteins containing single disulfide linkages by removing four native cysteine residues and replacing them with amino butyric acid. The ability for these constructs to directly anti-apoptotic Bcl-2 proteins in vitro was evaluated using fluorescence polarization. Our results reveal that one disulfide bond in particular, C8-C26, is highly influential for the proper folding of ScTx-Bax mimetics. Furthermore, the positioning of the disulfide seems to significantly affect the ability for ScTx-Bax proteins to target Bcl-2 in vitro. Two analogs: FluScTx-BaxΔΔ,8-26 (Kd = 377.6 nM) and FluScTx-BaxΔΔ,12-28 (Kd = 277.4 nM) were found to bind Bcl-2 with relatively high affinity, while a third: FluScTx-BaxΔΔ,3-21 (Kd = &gt;1,000 nM) did not bind Bcl-2 under these conditions.

Role of disulfide linkages in the folding and activity of scyllatoxin-based BH3 domain mimetics

254th National Meeting (2017)

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