Inhibitory Potential and Binding Thermodynamics of Scyllatoxin-Based BH3 Domain Mimetics Targeting Repressor BCL2 Proteins

Abstract

The B-cell lymphoma 2 (BCL2) proteins are a class of apoptosis regulators that control the release of apoptogenic factors from mitochondria. Under normal physiological conditions, apoptosis is inhibited through the actions of anti-apoptotic (repressor) BCL2 proteins that bind semi-indiscriminately to the helical BH3 domains of pro-apoptotic (effector) BCL2 proteins. In this work, we developed a series of BH3 domain mimetics by grafting residues from the effector BCL2 protein Bax onto the α-helix of scyllatoxin (ScTx). These so-called “ScTx-Bax” constructs were then used to gain insight into the physicochemical nature of repressor/effector BCL2 interactions. Specifically, we utilized competitive binding and isothermal titration calorimetry (ITC) to investigate the inhibitory potential and binding thermodynamics of ScTx-Bax structural variants that target the repressor protein Bcl-2 (proper) in vitro. Our data show that ScTx-Bax mimetics compete with isolated Bax BH3 domain peptides for Bcl-2 with IC₅₀ values in the mid-nanomolar range and that greater flexibility within the ScTx-Bax BH3 domain correlates with more effective inhibition. Furthermore, ITC experiments revealed that unstructured ScTx-Bax variants target Bcl-2 with greater entropic, but lower enthalpic, efficiencies than structured ScTx-Bax peptides. These results suggest that entropic contributions to binding Bcl-2 are more favorable for flexible BH3 domains; however, this enhancement is counterbalanced by a moderate enthalpic penalty. Overall, this study improves understanding of how structural properties of effector BH3 domains influence the promiscuous binding patterns of BCL2 proteins and expands the utility of ScTx-based BH3 domain mimetics as molecular tools to study discrete recognition elements that facilitate repressor/effector BCL2 interactions.